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Identification of antibodies targeting diverse functional epitopes on an antigen is highly crucial for discovering effective therapeutic candidates. Employing a traditional stepwise antibody "screening funnel" as well as prioritizing affinity-based selections over epitope-based selections, result in lead antibody panels lacking epitope diversity. In the present study, we employed an array-based surface plasmon resonance (SPR) platform to perform high-throughput epitope binning analysis on a large number of monoclonal antibodies (mAbs) generated in the early drug discovery process. The mAb panel contained clones from different antibody generation techniques and diverse transgenic mouse strains. The epitope binning results were analyzed in unique ways using various visualizations in the form of dendrograms and network plots, which assisted in determining diversity and redundancy in the mAb sample set. The binning data were further integrated with affinity information to evaluate the performance of seven different transgenic mouse strains. The combination of epitope binning results with binding kinetics and sequence analysis provided an effective and efficient way of selecting high affinity antibodies representing a diverse set of sequence families and epitopes.

Der Band thematisiert Theorien und Methoden zur Untersuchung der Kulturen regionaler Mehrsprachigkeit. Hierbei stehen das Verhältnis der Sprecher:innen zu ihren Regionalsprachen sowie deren Bewertung im mehrsprachigen Kontexten im Vordergrund. Die Beiträge leisten eine wissenschaftliche Einordnung der verschiedenen Gegenstandsbereiche der Kultur regionaler Mehrsprachigkeit. Dazu zeigen sie eine Vielzahl an Verfahren auf, wie diese theoretisch zu fassen und methodisch zu analysieren sind. Auf dieser Grundlage wird zu Beginn ein Überblicksmodell zur Sprachkultur erarbeitet. Der Ansatz des Bandes ist sprecher:innenzentriert, da die Perspektiven der Konzeptualisierung von Sprache, der Bewertung der Sprachkonstellationen sowie des Sprachgebrauchs den zentralen Fokus bilden.

International audience; Breton (Brittany, France) and Lower Sorbian (Brandenburg, Germany) are two of the many endangered minority languages currently undergoing revitalization. In their cases, given that intergenerational transmission in a family setting has mostly ceased, language revitalization takes the form of educational initiatives, such as the immersion program Diwan in Brittany and the bilingual program Witaj in Brandenburg. The article argues that the differences in language ideologies and attitudes of language revitalizers, which form the often unexpressed and unaddressed ideological foundations of these programs, have led to divergences in results of revitalization of Breton and Lower Sorbian. These divergences can have significant consequences for the new speakers of these languages produced through educational programs. Brittany's Diwan schools, which construes Breton as relevant to modern identities and capable of functioning outside the educational context, can be claimed to be producing new speakers in whose lives Breton will play a considerable role. By using foreign language teaching strategies and struggling to provide a vibrant life for Lower Sorbian after school, Witaj, in turn, does keep the minority language alive, but much more as a school subject than as a viable means of communication.

This paper explores the use of the Breton language (Brittany, North-West France) in contexts where speakers wish to signal their commitment to social equality through their linguistic practices. This is done with reference to examples of job advertisements which have pioneered the use of gender-fair language in Breton. Linguistic minorities are often portrayed as clinging to the past. This paper, however, sheds a different light on current minority language practices and demonstrates a progressive and egalitarian response to modernity among some current speakers of Breton, in their attempts to assume gender-fair stances.

Discourses which seek to position different speakers/users of Breton through the use of labels such as ‘traditional’, ‘new’, ‘learner’, ‘neo-bretonnant’, ‘brittophone’, etc. draw on persistent essentialist ideologies of language and create, in the process, contested elites and counter-elites in Breton-speaking networks. These discourses can be counter-productive towards projects which aim at producing multilingual citizens in Brittany at the present time. This article examines how stances of different speakers towards other speakers of Breton can involve jostling for positions of power within the Breton-speaking community and how attempts at creating elites and counter-elites seem to be a defining feature of contemporary revitalisation efforts in Brittany. This characterisation may, however, miss a ‘third space’ which some social actors may seek to engage with. These discourses are examined in this article through a critical sociolinguistic exploration of how (elite) multilingualism is constructed, maintained and contested by different actors in the Breton language community.

Differential Scanning Fluorimetry (DSF) is a method that enables rapid determination of a protein’s apparent melting temperature (Tma). Owing to its high throughput, DSF has found widespread application in fields ranging from structural biology to chemical screening. Yet DSF has developed two opposing reputations: one as an indispensable laboratory tool to probe protein stability, another as a frustrating platform that often fails. Here, we aim to reconcile these disparate reputations and help users perform more successful DSF experiments with three resources: an updated, interactive theoretical framework, practical tips, and online data analysis. We anticipate that these resources, made available online at DSFworld (https://gestwickilab.shinyapps.io/dsfworld/), will broaden the utility of DSF.

Educational initiatives in many minority language communities in Europe and beyond are producing ‘new’ speakers of the languages in question. The status of such speakers is often contested, however, and many people who have been through immersion education in a minority language can find themselves on the fringes of the language community of which their schooling was meant to make them ‘members’. This article explores the cases of ‘new’ speakers of Welsh in Wales and includes data in particular from the heartlands – West Wales – in which a number of ‘new’ speakers discuss their membership of Welsh-speaking communities, the difficulties they sometimes face and, crucially, how they manage to negotiate their own sense of speakerhood under such conditions. Also examined in this article are discourses on the same topic which appear in online blogs, and which would appear to point to a certain commonality of experience which is not confined to just those areas of Wales where Welsh is, or recently was, a community language, but is further echoed by new speakers in other parts of Wales outside the heartlands who have had similar experiences.

In many situations of minority language education, the focus has been on gains in the absolute numbers of speakers, with the result that less attention has been paid to the processes and linguistic outcomes associated with students in these educational programmes. In this article, we initiate a discussion on the revitalization situations in Brittany and Kashubia from a comparative perspective. In particular, we look at the different models of education in each of these regions and examine ethnographic data that highlight the attempts of students to attain legitimate ‘speakerhood’ of the minority languages in question. In particular, we take into the consideration the difficulties associated with these situations of attempted additive multilingualism when the general trend, among the majority populations, is toward standardized monolingualism. By way of a conclusion, we attempt to evaluate the different educational systems in both regions in terms of the production of future generations of ‘successful’ Kashubian and Breton speakers by examining the various language ideologies that are apparent in both situations of language revitalization.

Cysteine can be specifically functionalized by a myriad of acid-base conjugation strategies for applications ranging from probing protein function to antibody-drug conjugates and proteomics. In contrast, selective ligation to the other sulfur-containing amino acid, methionine, has been precluded by its intrinsically weaker nucleophilicity. Here, we report a strategy for chemoselective methionine bioconjugation through redox reactivity, using oxaziridine-based reagents to achieve highly selective, rapid, and robust methionine labeling under a range of biocompatible reaction conditions. We highlight the broad utility of this conjugation method to enable precise addition of payloads to proteins, synthesis of antibody-drug conjugates, and identification of hyperreactive methionine residues in whole proteomes.

This article examines some of the linguistic and ideological tensions resulting from language shift and subsequent revitalisation, using Breton as a case study. As a result of the opening up of ideological spaces in discourses on what it means to be a Breton speaker in the twenty-first century, the appearance of so-called ‘new’ speakers highlights a number of points of contestation. Operating within contexts which are becoming increasingly ‘postvernacular’ [Shandler, J. (2004). Postvernacular Yiddish: Language as a performance art. The Drama Review, 48(1), 19–43; Shandler, J. (2006). Adventures in Yiddishland: Postvernacular language and culture. Oakland: University of California Press] in nature, the use of Breton by these speakers can be viewed as more symbolic rather than communicative in many respects. Postvernacular use exists on a continuum of linguistic practice and vernacular use of Breton is still apparent, of course, and often indexed as the only ‘authentic’ and legitimate use of Breton....

For some years now, Welsh has been taught as a foreign language outside Wales, most especially in other Celtic countries, central Europe – and Poland. The first courses were established in the Catholic University of Lublin in the 1980s, and this provision has expanded over the years to include a Celtic language specialisation within the Faculty of English at Adam Mickiewicz University in Poznań. Relying at first on teachers from Wales to provide instruction in Welsh, the Centre for Celtic Studies is increasingly producing new, competent speakers/users of Welsh among the Poles. An obvious question to be asked concerns motivational issues – why, on the eastern edges of the European Union, are there people willing to put the effort into learning a language from the far west of Europe, when they have, in some cases, little contact with regular users of Welsh? Through the use of focus group interviews, the present study attempts to discover what motivates Polish students to study Welsh in a context of limited direct contact with the speakers of the language and limited, indirect access to Welsh language and culture.

Significance Next-generation sequencing (NGS) has allowed the comprehensive study of the genome and transcriptome. However, a similarly broad, highly multiplexed, and inexpensive method for proteomics using NGS remains elusive. Here, we describe a phage display-based method using preselected antibodies that are genetically encoded and capable of simultaneous profiling of hundreds of cell-surface targets on cells in culture or singly at low cost and without the need for chemical conjugation to purified antibodies. We use the method to identify cell-surface proteins that change in cancer cells, some of which are coordinately regulated and could lead to new biomarkers and cancer targets., Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states.

The introduction describes the field of material culture of multilingualism as a synthesis of two important provinces of human interest – ‘multilingualism’ and ‘material culture’. It starts with laying out some philosophical views on material culture from earlier times to the recent forays into the perception of materialities. The main thrust of the volume is the exposure of natural ties between language, cognition, identity and the material world. A special section of this chapter traces the gradual realization of applied linguists of the importance of ‘things’ and ‘places’ for the construction of meaning and communication. In putting forward the material culture of multilingualism as a distinct domain of scholarly interest, the author-editors call for encompassing the entire material culture realm, that is, the whole environment of multilinguals. They argue for going beyond geographical placement, to include private places and in-between spaces, in addition to public ones, traditionally considered with regard to language and for appreciating features of material culture as dynamic, changeable in time, space, form, and value thus linking languages with the physical environment where they are used. This introduction also contains a description of the rationale, scope and the objective of the volume, and gives a brief overview of the contributions in the volume.

Minority cultures and associated efforts to maintain and promote their linguistic varieties in situations of unbalanced multilingualism face many challenges. Pressure to adapt to modernity and globalization mean that cultural and linguistic minorities are having to find new and creative ways to express their identities and collective sense of community. This article examines the situation of the Lemko language in Poland, estimated to be spoken by around 11,000 people (Przynaleznośc narodowo-etniczna ludności, GUS. 2011. Material na konferencje prasową w dniu 29. 01. 2013. p 3. Accessed on 6 Mar 2013). Educational programmes, such as the Russian-with-Lemko degree taught at the University of Cracow, or the teaching of Lemko in selected schools in the traditional Lemko areas, are complemented by events and festivals which might equip younger generations of speakers of the language with the cultural material they require in order to achieve ‘authentic speakerhood’. This concept will be examined in the present article, drawing on fieldwork undertaken in 2012 and 2013 at a number of sites in Poland, and linking it to the concept of the “material culture of multilingualism” (Aronin L. Stud Second Lang Learn Teach 2(2):79–191, 2012) in order to explore the role cultural and semiotic artefacts play in the construction of a Lemko multilingual identity in the twenty-first century.

This article analyzes the tensions and dynamics which exist between “new” speakers and other speakers, such as traditional or native speakers of minority languages (MLs), in an attempt to discover just how much of a barrier to communication are the (perceived) differences which are purported to exist between them. The dynamics between “new” and native speakers seem to be complex and nuanced, and “(in)authenticity” can be indexed through accent, the lexicon and grammatical structures, both by local users and more widely by researchers and other interested third parties, reflecting a wide range of ideo-logical stances. Using a critical sociolinguistic framework, these differences are examined from the perspective of the power differentials among and between various ML speakers/users in two situations of language endangerment, Breton and Yiddish. The reproduction of “symbolic violence”, as described by Bourdieu (1991), which results from such differentials can hinder language revitalization projects and can run counter to the interests of the language community in question. Both settings appear to share a commonality of experience that is wider than just the two language communities under scrutiny here and possible ways of reconciling such differences are examined toward the end of the article.

Heterochromatin protein 1 (HP1) family proteins are conserved chromatin binding proteins involved in gene silencing, chromosome packaging, and chromosome segregation. These proteins recognize histone H3 lysine 9 methylated tails via their chromodomain and recruit additional ligand proteins with diverse activities through their dimerization domain, the chromoshadow domain. Species that have HP1 proteins possess multiple paralogs that perform non-overlapping roles in vivo. How different HP1 proteins, which are highly conserved, perform different functions is not well understood. Here, we use the two Schizosaccharomyces pombe HP1 paralogs, Swi6 and Chp2, as model systems to compare and contrast their biophysical properties. We find that Swi6 and Chp2 have similar dimerization and oligomerization equilibria, and that Swi6 binds slightly (~3-fold) more strongly to nucleosomes than Chp2. Furthermore, while Swi6 binding to the H3K9me3 mark is regulated by a previously described auto-inhibition mechanism, the binding of Chp2 to the H3K9me3 mark is not analogously regulated. In the context of chromoshadow domain interactions, we show using a newly identified peptide sequence from the Clr3 histone deacetylase and a previously identified sequence from the protein Shugoshin that the Swi6 chromoshadow domain binds both ligands more strongly than the Chp2. Overall, our findings uncover quantitative differences in how Swi6 and Chp2 interact with nucleosomal and non-nucleosomal ligands and qualitative differences in how their assembly on nucleosomes is regulated. These findings provide a biochemical framework to explain the varied functions of Chp2 and Swi6 in vivo.

Lemko identity in Poland is contested in a number of contexts, including social, linguistic and political domains, among others. The members of this minority have to learn to negotiate multiple identities, not only from an in-group perspective but also in interactions with the majority community in Poland. This paper examines how the Lemkos attempt to do this, with varying degrees of success, and the tensions which arise as a result. In particular, a Lemko identity is examined from the perspective of ideologies of language in order to draw out the major themes which are apparent in this re-emergent minority.

High-quality immunoreagents enhance the performance and reproducibility of immunoassays and, in turn, the quality of both biological and clinical measurements. High quality recombinant immunoreagents are generated using antibody-phage display. One metric of antibody quality – the binding affinity – is quantified through the dissociation constant (KD) of each recombinant antibody and the target antigen. To characterize the KD of recombinant antibodies and target antigen, we introduce affinity electrophoretic mobility shift assays (EMSAs) in a high-throughput format suitable for small volume samples. A microfluidic card comprised of free-standing polyacrylamide gel (fsPAG) separation lanes supports 384 concurrent EMSAs in 30 s using a single power source. Sample is dispensed onto the microfluidic EMSA card by acoustic droplet ejection (ADE), which reduces EMSA variability compared to sample dispensing using manual or pin tools. The KD for each of a six-member fragment antigen-binding fragment library is reported using ~25-fold less sample mass and ~5-fold less time than conventional heterogeneous assays. Given the form factor and performance of this micro- and mesofluidic workflow, we have developed a sample-sparing, high-throughput, solution-phase alternative for biomolecular affinity characterization.

Ile jest dzisiaj języków na świecie? Liczba podana w naszej książce może być zaskakująca, zwłaszcza, że jest ona kilkunastokrotnie wyższa niż liczba państw na świecie. Języki również cały czas powstają – czy to za sprawą indywidualnego rozwoju dialektów danego języka czy też mieszania się różnych języków w społeczeństwach wielojęzycznych. Niestety, języki o wiele częściej zanikają niż powstają, co powoduje, że ogólna liczba języków wcale nie wzrasta, a wręcz dramatycznie maleje. Trudno przewidzieć, ile języków będzie nadal używanych za kilkanaście - kilkadziesiąt lat, jednak wstępne prognozy są pesymistyczne: połowa z obecnie używanych języków umrze. Nasze języki są w niebezpieczeństwie i nie jest to sprawa, którą interesują się wyłącznie językoznawcy. Różnorodność językowa to część różnorodności kulturowej, która cechuje człowieczeństwo – i jest ona skarbem, a nie ciężarem. Z pomocą tej książki dowiesz się, co niesie ze sobą ten skarb: fascynujący świat języków.

Prostasin or human channel-activating protease 1 has been reported to play a critical role in the regulation of extracellular sodium ion transport via its activation of the epithelial cell sodium channel. Here, the structure of the extracellular portion of the membrane associated serine protease has been solved to high resolution in complex with a nonselective d-FFR chloromethyl ketone inhibitor, in an apo form, in a form where the apo crystal has been soaked with the covalent inhibitor camostat and in complex with the protein inhibitor aprotinin. It was also crystallized in the presence of the divalent cation Ca(+2). Comparison of the structures with each other and with other members of the trypsin-like serine protease family reveals unique structural features of prostasin and a large degree of conformational variation within specificity determining loops. Of particular interest is the S1 subsite loop which opens and closes in response to basic residues or divalent ions, directly binding Ca(+2) cations. This induced fit active site provides a new possible mode of regulation of trypsin-like proteases adapted in particular to extracellular regions with variable ionic concentrations such as the outer membrane layer of the epithelial cell.

Globalisation processes, often accused of imposing uniformity the world over, also open up opportunities for minorities (including linguistic ones) to exploit a variety of spaces for self-definition and self-expression. The expansion in the use of minority languages in public signage is one consequence of this present phase of reorientation in normative space. On one level, the appearance of the Breton language in more and more public places in Brittany has been welcomed by language activists as a step forward, but on another level, such tokenism has not necessarily led to an expansion in the public use of the language on a more informal basis. This paper explores the effect of the symbolic use of the language on young Breton speakers being educated in immersion and bilingual schools in one town in Brittany and the dynamic that results from their encountering Breton in one establishment that has come to encapsulate ‘High Modernity’ the world over.

The substrate activity screening (SAS) method, a substrate-based fragment identification and optimization method for the development of enzyme inhibitors, was previously applied to cathepsin S to obtain a novel (2-arylphenoxy)acetaldehyde inhibitor, 2, with a 0.49 microM Ki value (Wood, W. J. L.; Patterson, A. W.; Tsuruoka, H.; Jain, R. K.; Ellman, J. A. J. Am. Chem. Soc. 2005, 127, 15521-15527). In this paper we disclose the X-ray structure of a complex between cathepsin S and inhibitor 2 which reveals an unprecedented binding mode. On the basis of this structure, additional 2-biaryloxy substrates with greatly increased cleavage efficiency were designed. Conversion of the optimized substrates to the corresponding aldehyde inhibitors yielded a low molecular weight (304 Daltons) and potent (9.6 nM) cathepsin S inhibitor that showed from 100- to >1000-fold selectivity relative to cathepsins B, L, and K.

Antibodies are key reagents in biology and medicine, but commercial sources are rarely recombinant and thus do not provide a permanent and renewable resource. Here, we describe an industrialized platform to generate antigens and validated recombinant antibodies for 346 transcription factors (TFs) and 211 epigenetic antigens. We describe an optimized automated phage display and antigen expression pipeline that in aggregate produced about 3000 sequenced Fragment antigen-binding domain that had high affinity (typically EC50

The Recombinant Antibody Network (RAN) is a pilot project involving 3 integrated automation centres to create renewable, open-source, high-quality binding reagents. The main aims of RAN are to: gen...

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

Interest in the disappearance of languages has developed to a point where language death and obsolescence can be viewed as a field in its own right. Recent academic publications on language endangerment, that is, the process whereby languages lose ground to other languages either because they are spoken by fewer and fewer speakers and/or because they are heavily influenced by one or more competing languages, include Crystal (2000), Dalby (2002), Harrison (2007), Maffi (2001), Nettle and Romaine (2000), Phillipson (2001) and Skutnabb-Kangas (2000). These authors basically share the same positions, though there are minor differences in the specific ways they state them. This interest is often accompanied by a ‘discourse of language endangerment’, which has been described by Dobrin et al. (2007: 59) as ‘an ideologically charged discursive space that is kept bustling by a number of overlapping constituencies’, such as linguists, minority activists, international groups, funding institutions, conservation groups and the media.