Combining Oligo Pools and Golden Gate Cloning to Create Protein Variant Libraries or Guide RNA Libraries for CRISPR Applications.

Oligo pools are array-synthesized, user-defined mixtures of single-stranded oligonucleotides that can be used as a source of synthetic DNA for library cloning. While currently offering the most affordable source of synthetic DNA, oligo pools also come with limitations such as a maximum synthesis length (approximately 350 bases), a higher error rate compared to alternative synthesis methods, and the presence of truncated molecules in the pool due to incomplete synthesis. Here, we provide users with a comprehensive protocol that details how oligo pools can be used in combination with Golden Gate cloning to create user-defined protein mutant libraries, as well as single-guide RNA libraries for CRISPR applications. Our methods are optimized to work within the Yeast Toolkit Golden Gate scheme, but are in principle compatible with any other Golden Gate-based modular cloning toolkit and extendable to other restriction enzyme-based cloning methods beyond Golden Gate. Our methods yield high-quality, affordable, in-house variant libraries.

Advancements in mammalian display technology for therapeutic antibody development and beyond: current landscape, challenges, and future prospects.

The evolving development landscape of biotherapeutics and their growing complexity from simple antibodies into bi- and multi-specific molecules necessitates sophisticated discovery and engineering platforms. This review focuses on mammalian display technology as a potential solution to the pressing challenges in biotherapeutic development. We provide a comparative analysis with established methodologies, highlighting key aspects of mammalian display technology, including genetic engineering, construction of display libraries, and its pivotal role in hit selection and/or developability engineering. The review delves into the mechanisms underpinning developability-driven selection via mammalian display and their broader implications. Applications beyond antibody discovery are also explored, alongside advancements towards function-first screening technologies, precision genome engineering and AI/ML-enhanced libraries, situating them in the context of mammalian display. Overall, the review provides a comprehensive overview of the current mammalian display technology landscape, underscores the expansive potential of the technology for biotherapeutic development, addresses the critical challenges for the full realisation of this potential, and examines advances in related disciplines that might impact the future application of mammalian display technologies.

Electrochemical C-O and C-N Arylation using Alternating Polarity in Flow for Compound Libraries.

Etherification and amination of aryl halide scaffolds are commonly used reactions in parallel medicinal chemistry to rapidly scan structure-activity relationships with abundant building blocks. Electrochemical methods for aryl etherification and amination demonstrate broad functional group tolerance and extended nucleophile scope compared to traditional methods. Nevertheless, there is a need for robust and scale-transferable workflows for electrochemical compound library synthesis. Herein we describe a platform for automated electrochemical synthesis of C-X arylation (X = NH, OH) in flow to access compound libraries. A comprehensive Design of Experiment (DoE) study identifies an optimal protocol which generates high yields across > 30 aryl halide scaffolds, diverse amines (including electron-deficient sulfonamides, sulfoximines, amides, and anilines) and alcohols (including serine residues within peptides). Reaction sequences are automated on commercially available equipment to generate libraries of anilines and aryl ethers. The unprecedented application of potentiostatic alternating polarity in flow is essential to avoid accumulating electrode passivation. Moreover, it enables reactions to be performed in air, without supporting electrolyte and with high reproducibility over consecutive runs. Our method represents a powerful means to rapidly generate nucleophile independent C-X arylation compound libraries using flow electrochemistry.

Identification of organic contaminants in water and related matrices using untargeted liquid chromatography high-resolution mass spectrometry screening with MS/MS libraries.

Nontargeted and suspect screening with liquid chromatography-high resolution mass spectrometry (LC-HRMS) has become an indispensable tool for quality assessment in the aquatic environment - complementary to targeted analysis of organic (micro)contaminants. An LC-HRMS method is presented, suitable for the analysis of a wide variety of water related matrices: surface water, groundwater, wastewater, sediment and sludge, including extracts from passive samplers and on-site solid phase enrichment, while focusing on the data processing aspect of the method. A field study is included to demonstrate the practical application and versatility of the whole process. HRMS/MS data were recorded following LC separation in both (ESI) positive and negative ionization modes using data dependent as well as data independent acquisition. Two vendor (Agilent's Personal Compound Database and Library and from National Institute of Standards and Technology) and one open (MassBank/EU) tandem mass spectral libraries were utilized for the identification of compounds via mass spectral match. The development of a novel software tool for parsing, grouping and reduction of MS/MS features in data files converted to mascot generic format (MGF) helped to substantially decrease the amount of time and effort needed for MS library search. While applying the method, in the course of the entire field study, 18771 detections (from 870 individual compounds) in total were recorded in 275 samples, resulting in 68.3 identified compounds per sample, on average. Among the top ten most frequently detected contaminants across all samples and sample types were pharmaceutical compounds carbamazepine, 4-acetamidoantipyrine, 4-formylaminoantipyrine, tramadol, lamotrigine and phenazone and industrial contaminants toluene-2-sulfonamide, tolytriazole, tris(2-butoxyethyl) phosphate and benzotriazole. Exploratory data analysis methods and tools enabled us to discover organic pollutant occurrence patterns within the comprehensive sets of qualitative data collected from various projects between the years 2018-2023. The results may be used as valuable inputs for future water quality monitoring programs.

Developing a foundation information and academic skills programme for potential Student Nursing Associates.

This article outlines the development of 'Prepare to Progress', a preapplication programme for potential Student Nursing Associate (SNA) applicants at Gloucestershire Hospitals NHS Foundation Trust. Created collaboratively by the Trust's Library and Knowledge Services and Professional Education teams, the programme aims to provide realistic course expectations, teach study skills and boost confidence in using library services. Evaluation results indicate increased understanding of the SNA course, improved application decision-making, and enhanced academic confidence among participants. The programme demonstrates the valuable role of library services in preparing healthcare support workers for further education and addressing library anxiety. The study suggests benefits for both participants and library services.

Encoding and display technologies for combinatorial libraries in drug discovery: The coming of age from biology to therapy.

Drug discovery is a sophisticated process that incorporates scientific innovations and cutting-edge technologies. Compared to traditional bioactivity-based screening methods, encoding and display technologies for combinatorial libraries have recently advanced from proof-of-principle experiments to promising tools for pharmaceutical hit discovery due to their high screening efficiency, throughput, and resource minimization. This review systematically summarizes the development history, typology, and prospective applications of encoding and displayed technologies, including phage display, ribosomal display, mRNA display, yeast cell display, one-bead one-compound, DNA-encoded, peptide nucleic acid-encoded, and new peptide-encoded technologies, and examples of preclinical and clinical translation. We discuss the progress of novel targeted therapeutic agents, covering a spectrum from small-molecule inhibitors and nonpeptidic macrocycles to linear, monocyclic, and bicyclic peptides, in addition to antibodies. We also address the pending challenges and future prospects of drug discovery, including the size of screening libraries, advantages and disadvantages of the technology, clinical translational potential, and market space. This review is intended to establish a comprehensive high-throughput drug discovery strategy for scientific researchers and clinical drug developers.

GGAssembler: Precise and economical design and synthesis of combinatorial mutation libraries.

Golden Gate assembly (GGA) can seamlessly generate full-length genes from DNA fragments. In principle, GGA could be used to design combinatorial mutation libraries for protein engineering, but creating accurate, complex, and cost-effective libraries has been challenging. We present GGAssembler, a graph-theoretical method for economical design of DNA fragments that assemble a combinatorial library that encodes any desired diversity. We used GGAssembler for one-pot in vitro assembly of camelid antibody libraries comprising >105 variants with DNA costs <0.007$ per variant and dropping significantly with increased library complexity. >93% of the desired variants were present in the assembly product and >99% were represented within the expected order of magnitude as verified by deep sequencing. The GGAssembler workflow is, therefore, an accurate approach for generating complex variant libraries that may drastically reduce costs and accelerate discovery and optimization of antibodies, enzymes and other proteins. The workflow is accessible through a Google Colab notebook at https://github.com/Fleishman-Lab/GGAssembler.

Involvement of MicroRNAs in the Hypersensitive Response of Capsicum Plants to the Capsicum Chlorosis Virus at Elevated Temperatures.

The orthotospovirus capsicum chlorosis virus (CaCV) is an important pathogen affecting capsicum plants. Elevated temperatures may affect disease progression and pose a potential challenge to capsicum production. To date, CaCV-resistant capsicum breeding lines have been established; however, the impact of an elevated temperature of 35 °C on this genetic resistance remains unexplored. Thus, this study aimed to investigate how high temperature (HT) influences the response of CaCV-resistant capsicum to the virus. Phenotypic analysis revealed a compromised resistance in capsicum plants grown at HT, with systemic necrotic spots appearing in 8 out of 14 CaCV-infected plants. Molecular analysis through next-generation sequencing identified 105 known and 83 novel microRNAs (miRNAs) in CaCV-resistant capsicum plants. Gene ontology revealed that phenylpropanoid and lignin metabolic processes, regulated by Can-miR408a and Can- miR397, are likely involved in elevated-temperature-mediated resistance-breaking responses. Additionally, real-time PCR validated an upregulation of Can-miR408a and Can-miR397 by CaCV infection at HT; however, only the Laccase 4 transcript, targeted by Can-miR397, showed a tendency of negative correlation with this miRNA. Overall, this study provides the first molecular insights into how elevated temperature affects CaCV resistance in capsicum plants and reveals the potential role of miRNA in temperature-sensitive tospovirus resistance.

Construction and characterization of DNA libraries from cultured phages and environmental viromes.

Despite many efforts to understand and leverage the functional potential of environmental viromes, most bacteriophage genes are largely uncharacterized. To explore novel biology from uncultivated microbes like phages, metagenomics has emerged as a powerful tool to directly mine new genes without the need to culture the diverse microbiota and the viruses within. When a pure computational approach cannot infer gene function, it may be necessary to create a DNA library from environmental genomic DNA, followed by the screening of that library for a particular function. However, these screens are often initiated without a metagenomic analysis of the completed DNA library being reported. Here, we describe the construction and characterization of DNA libraries from a single cultured phage (ΦT4), five cultured Escherichia coli phages, and three metagenomic viral sets built from freshwater, seawater, and wastewater samples. Through next-generation sequencing of five independent samplings of the libraries, we found a consistent number of recovered genes per replicate for each library, with many genes classifiable via the KEGG and Pharokka databases. By characterizing the size of the genes and inserts, we found that our libraries contain a median of one to two genes per contig with a median gene length of 303-381 bp for all libraries, reflective of the small genomes of viruses. The environmental libraries were genetically diverse compared to the single phage and multi-phage libraries. Additionally, we found reduced coverage of individual genomes when five phages were used as opposed to one. Taken together, this work provides a comprehensive analysis of the DNA libraries from phage genomes that can be used for metagenomic exploration and functional screens to infer and identify new biology.IMPORTANCEFunctional metagenomics is an approach that aims to characterize the putative biological function of genes in the microbial world. This includes an examination of the sequencing data collected from a pooled source of diverse microbes and inference of gene function by comparison to annotated and studied genes from public databases. At times, DNA libraries are made from these genes, and the library is screened for a specific function. Hits are validated using a combination of biological, computational, and structural analysis. Left unresolved is a detailed characterization of the library, both its diversity and content, for the purposes of imputing function entirely by computational means, a process that may yield findings that aid in designing useful screens to identify novel gene functions. In this study, we constructed libraries from cultured phages and uncultured viromes from the environment and characterized some important parameters, such as gene number, genes per contig, ratio of hypothetical to known proteins, total genomic coverage and recovery, and the effect of pooling genetic information from multiple sources, to provide a better understanding of the nature of these libraries. This work will aid the design and implementation of future screens of pooled DNA libraries to discover and isolate viral genes with novel biology across various biomes.

Construction and validation of a synthetic phage-displayed nanobody library.

Nanobodies derived from camelids and sharks offer unique advantages in therapeutic applications due to their ability to bind to epitopes that were previously inaccessible. Traditional methods of nanobody development face challenges such as ethical concerns and antigen toxicity. Our study presents a synthetic, phagedisplayed nanobody library using trinucleotide-directed mutagenesis technology, which allows precise amino acid composition in complementarity-determining regions (CDRs), with a focus on CDR3 diversity. This approach avoids common problems such as frameshift mutations and stop codon insertions associated with other synthetic antibody library construction methods. By analyzing FDA-approved nanobodies and Protein Data Bank sequences, we designed sub-libraries with different CDR3 lengths and introduced amino acid substitutions to improve solubility. The validation of our library through the successful isolation of nanobodies against targets such as PD-1, ATXN1 and STAT3 demonstrates a versatile and ethical platform for the development of high specificity and affinity nanobodies and represents a significant advance in biotechnology.

The Discovery of a Specific CKIP-1 Ligand for the Potential Treatment of Disuse Osteoporosis.

Bone homeostasis relies on the delicate balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption. The casein kinase 2 interacting protein-1 (CKIP-1), a specific CK2α subunit-interacting protein, has been documented as one of the crucial negative regulators of bone formation. CKIP-1 siRNA therapy has constraints that limit its use in clinical applications. Therefore, it is necessary to explore effective targeting strategies for CKIP-1. In this study, we observed an upregulation of CKIP-1 protein expression in the microgravity environment, while its ubiquitination levels decreased. We further investigated the interaction between CKIP-1 and VHL and found that VHL enhanced CKIP-1 degradation through the ubiquitylation-proteasome system (UPS). Additionally, we discovered a small molecule ligand, named C77, through DNA-encoded library (DEL) screening, which binds to CKIP-1 both in vivo and in vitro, as confirmed by Surface Plasmon Resonance (SPR) and the Cellular Thermal shift assay (CETSA), respectively. Our findings demonstrated the potential of VHL and C77 as guiding factors in the development of CKIP-1-based Proteolysis-Targeting Chimeras (PROTACs), which could be future therapeutic interventions in disuse osteoporosis.

Evolving from public health libraries as a place to focus on public health librarian expertise.

Objective: This article describes the evolution of academic public health library services from standalone academic public health libraries in 2004 to centralized services by 2021. Methods: Five public health libraries serving public health graduate programs (SPH) at public and private institutions were visited in 2006-07. Visits comprised tours, semi-structured interviews with librarians and local health department staff, and collecting of contemporary print documents. We compiled and compared visit notes across libraries. In 2022, we reviewed online materials announcing library closure or transition for timing and how services were to be subsequently provided. Results: Libraries and SPH were co-located and most librarians maintained public health expertise though they did not have faculty appointments in their SPHs. Specialized statistical and geographic information systems (GIS) software and data were provided in partnership, often with other system libraries. Only two libraries had strong connections to health departments-one with direct service agreements and another engaged in public health training. Conclusion: Academic public health libraries' relationships with SPHs and health departments did not ensure their existence as standalone entities. Following a national trend for branch libraries, public health information services were centralized into larger health or science libraries. The scope and specialization of librarian expertise continues to be valued with several institutions having librarians dedicated to public health.

Health sciences faculty publication patterns and related information-seeking behavior.

Objectives: This study aims to explore how health science faculty publication patterns at a large public research university have changed over time and examine how productivity relates to their information-seeking behavior and perception of the academic library. Methods: Two datasets were utilized: one consisted of publication records of health sciences faculty spanning a 15-year period, while the other was from a faculty survey exploring faculty's perception of and satisfaction with library resources and services related to their research. Results: Health sciences faculty publication patterns have changed over time, characterized by greater productivity, collaboration, and use of literature in their publications. Faculty's literature use correlates with productivity, as evidenced by both datasets. The survey revealed that faculty with more publications tend to rely more on online journals and Interlibrary Loan (ILL). Similarly, the publication data indicated that less productive faculty tended to use fewer references in their publications. Discussion: The publication data and survey results offer valuable insights into the health sciences faculty's information-seeking behavior and productivity. Online access to information has been effective in facilitating use of information, as indicated by the greater incorporation of references in publications. Conclusion: The study highlights the changing publication patterns and productivity of health sciences faculty, as well as the role academic libraries play in supporting their research and publishing activities. Although multiple variables influence faculty access to and use of information, faculty attitudes towards the library and use of the library are related to faculty research and productivity.

Promoting nutrition literacy in children: a case study of a community partnership between a university and an elementary school.

Background: Health literacy outreach is commonplace within public and hospital libraries but less so in academic libraries, where it is often viewed as not integral. Academic health science libraries may collaborate with public libraries to provide public health information literacy programming or "train the trainer" sessions, but examples of academic health science librarians leading community health initiatives are still limited. Case Presentation: This case report discusses a collaborative project between Gonzaga's Foley Center Library, the School of Nursing and Human Physiology, and a local elementary school to promote health literacy for students and their families, led by an Academic Health Sciences Librarian. The project scope included delivering nutrition education to elementary school students and their families, but pandemic closures limited plans for in-person programming. Conversations with stakeholders led to additional project opportunities, including tabling at the local block party, collaborating on a campus visit for 5th and 6th graders, supporting middle school cooking classes, and the creation of a toolkit for elementary and middle school teachers to support curriculum about healthy body image and potential disordered eating. Conclusion: This project demonstrates one example of how academic libraries can partner with other campus departments to support health literacy outreach in their local communities. The pandemic made planning for in-person programming tenuous, but by expanding meetings to include staff from other areas of the university, the project team was able to tap into additional outreach opportunities. This work fostered close relationships with the local elementary school, providing the groundwork for collaborative health programming in the future, though more thorough assessment is suggested for future projects.

Natural Products Dereplication: Databases and Analytical Methods.

The development of efficient methods for dereplication has been critical in the re-emergence of the research in natural products as a source of drug leads. Current dereplication workflows rapidly identify already known bioactive secondary metabolites in the early stages of any drug discovery screening campaign based on natural extracts or enriched fractions. Two main factors have driven the evolution of natural products dereplication over the last decades. First, the availability of both commercial and public large databases of natural products containing the key annotations against which the biological and chemical data derived from the studied sample are searched for. Second, the considerable improvement achieved in analytical technologies (including instrumentation and software tools) employed to obtain robust and precise chemical information (particularly spectroscopic signatures) on the compounds present in the bioactive natural product samples. This chapter describes the main methods of dereplication, which rely on the combined use of large natural product databases and spectral libraries, alongside the information obtained from chromatographic, UV-Vis, MS, and NMR spectroscopic analyses of the samples of interest.

Fish species authentication in commercial fish products using mass spectrometry and spectral library matching approach.

Seafood fraud has become a global issue, threatening food security and safety. Adulteration, substitution, dilution, and incorrect labeling of seafood products are fraudulent practices that violate consumer safety. In this context, developing sensitive, robust, and high-throughput molecular tools for food and feed authentication is becoming crucial for regulatory purposes. Analytical approaches such as proteomics mass spectrometry have shown promise in detecting incorrectly labeled products. For the application of these tools, genome information is crucial, but currently, for many marine species of commercial importance, such information is unavailable. However, when combining proteomic analysis with spectral library matching, commercially important fish species were successfully identified, differentiated, and quantified in pure muscle samples and mixtures, even when genome information was scarce. This study further tested the previously developed spectral library matching approach to differentiate between 29 fish species from the North Sea and examined samples including individual fish, laboratory-prepared mixtures and commercial products. For authenticating libraries generated from 29 fish species, fresh muscle samples from the fish samples were matched against the reference spectral libraries. Species of the fresh fish samples were correctly authenticated using the spectral library approach. The same result was obtained when evaluating the laboratory-prepared mixtures. Furthermore, processed commercial products containing mixtures of two or three fish species were matched against these reference spectral libraries to test the accuracy and robustness of this method for authentication of fish species. The results indicated that the method is suitable for the authentication of fish species from highly processed samples such as fish cakes and burgers. The study shows that current and future challenges in food and feed authentication can efficiently be tackled by reference spectral libraries method when prospecting new resources in the Arctic.

Core collections: Essential titles for health libraries.

Core collections have been produced by CILIP's Health Libraries Group, then called the Library Association's Medical Section, since 1952. Maintained by a Working Group of health librarians based in the UK NHS, higher education and specialist libraries, the collections provide an up-to-date curated list of reliable titles essential to health libraries. The core collections currently include nursing, midwifery, medicine and dentistry. The newest core collection is being developed in collaboration with the African Hospital Libraries to provide a list of key resources relevant to sub-Saharan Africa. Expressions of interest to help develop this latest collection are invited.

Perspectives on current approaches to virtual screening in drug discovery.

INTRODUCTION: For the past two decades, virtual screening (VS) has been an efficient hit finding approach for drug discovery. Today, billions of commercially accessible compounds are routinely screened, and many successful examples of VS have been reported. VS methods continue to evolve, including machine learning and physics-based methods. AREAS COVERED: The authors examine recent examples of VS in drug discovery and discuss prospective hit finding results from the critical assessment of computational hit-finding experiments (CACHE) challenge. The authors also highlight the cost considerations and open-source options for conducting VS and examine chemical space coverage and library selections for VS. EXPERT OPINION: The advancement of sophisticated VS approaches, including the use of machine learning techniques and increased computer resources as well as the ease of access to synthetically available chemical spaces, and commercial and open-source VS platforms allow for interrogating ultra-large libraries (ULL) of billions of molecules. An impressive number of prospective ULL VS campaigns have generated potent and structurally novel hits across many target classes. Nonetheless, many successful contemporary VS approaches still use considerably smaller focused libraries. This apparent dichotomy illustrates that VS is best conducted in a fit-for-purpose way choosing an appropriate chemical space. Better methods need to be developed to tackle more challenging targets.

A next-generation Fab library platform directly yielding drug-like antibodies with high affinity, diversity, and developability.

We previously described an in vitro single-chain fragment (scFv) library platform originally designed to generate antibodies with excellent developability properties. The platform design was based on the use of clinical antibodies as scaffolds into which replicated natural complementarity-determining regions purged of sequence liabilities were inserted, and the use of phage and yeast display to carry out antibody selection. In addition to being developable, antibodies generated using our platform were extremely diverse, with most campaigns yielding sub-nanomolar binders. Here, we describe a platform advancement that incorporates Fab phage display followed by single-chain antibody-binding fragment Fab (scFab) yeast display. The scFab single-gene format provides balanced expression of light and heavy chains, with enhanced conversion to IgG, thereby combining the advantages of scFvs and Fabs. A meticulously engineered, quality-controlled Fab phage library was created using design principles similar to those used to create the scFv library. A diverse panel of binding scFabs, with high conversion efficiency to IgG, was isolated against two targets. This study highlights the compatibility of phage and yeast display with a Fab semi-synthetic library design, offering an efficient approach to generate drug-like antibodies directly, facilitating their conversion to potential therapeutic candidates.

Help-Seeking Situations Related to Visual Interactions on Mobile Platforms and Recommended Designs for Blind and Visually Impaired Users.

While it is common for blind and visually impaired (BVI) users to use mobile devices to search for information, little research has explored the accessibility issues they encounter in their interactions with information retrieval systems, in particular digital libraries (DLs). This study represents one of the most comprehensive research projects, investigating accessibility issues, especially help-seeking situations BVI users face in their DL search processes. One hundred and twenty BVI users were recruited to search for information in six DLs on four types of mobile devices (iPhone, iPad, Android phone, and Android tablet), and multiple data collection methods were employed: questionnaires, think-aloud protocols, transaction logs, and interviews. This paper reports part of a large-scale study, including the categories of help-seeking situations BVI users face in their interactions with DLs, focusing on seven types of help-seeking situations related to visual interactions on mobile platforms: difficulty finding a toggle-based search feature, difficulty understanding a video feature, difficulty navigating items on paginated sections, difficulty distinguishing collection labels from thumbnails, difficulty recognizing the content of images, difficulty recognizing the content of graphs, and difficulty interacting with multilayered windows. Moreover, corresponding design recommendations are also proposed: placing meaningful labels for icon-based features in an easy-to-access location, offering intuitive and informative video descriptions for video players, providing structure information about a paginated section, separating collection/item titles from thumbnail descriptions, incorporating artificial intelligence image/graph recognition mechanisms, and limiting screen reader interactions to active windows. Additionally, the limitations of the study and future research are discussed.