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.

Fragment and torsion biasing algorithms for construction of small organic molecules in proteins using DOCK.

The computational construction of small organic molecules (de novo design), directly in a protein binding site, is an effective means for generating novel ligands tailored to fit the pocket environment. In this work, we present two new methods, which aim to improve de novo design outcomes using (1) biasing algorithms to prioritize selection and/or acceptance of fragments and torsions during growth, and (2) parallel-based clustering and pruning algorithms to remove duplicate molecules as candidate fragment are added. Large-scale testing encompassing thousands of simulations were employed to interrogate the methods in terms of multiple metrics which include numbers of duplicate molecules generated, pairwise-similarity, focused library reconstruction rates, fragment and torsion frequencies, fragment and torsion rank scores, interaction energy and drug-likeness scores, and 3D pose comparisons. The biasing algorithms, particularly those that include fragment and torsion components simultaneously, led to molecules that more closely mimicked the distributions of fragments and torsions found in drug-like libraries. The new parallel-based clustering and pruning algorithms, compared with the existing serial approach, also led to larger ensembles comprised of topologically unique molecules with much greater efficiency by removing redundant growth paths.

A community engagement program to improve awareness for credible online health information.

Background: The volume of online health information available makes it difficult to navigate and check its validity and reliability. A community-based MedlinePlus training program was developed to improve participants' ability to access credible online health information. Case Presentation: The program was a public-private partnership between a managed care organization and four local public libraries. A total of eight programs were held between October and November 2017. Each program had a 30-minute cooking demonstration followed by a 30-minute training on access to and navigation of the MedlinePlus website. Program participants were Medicaid beneficiaries, dually eligible for Medicare and Medicaid beneficiaries, and community members from a Pennsylvania county (n=39). A pre-and post-training questionnaire was administered to assess participants' knowledge and practice, and their ability to access health information on the MedlinePlus website. We conducted a retrospective analysis of the data collected during the MedlinePlus trainings. Results from the Wilcoxon Signed Rank test indicated no statistically significant change in participants' ability to access information (Z= -1.41, p=0.16) after attending the program. Conclusion: Although the median pre- to post-program responses improved from 'incorrect' to 'correct,' the number of programs held, and low attendance might be the reason for non-significant results. Participants reported that the program was informative, the website was comprehensive and user-friendly, and they were impressed by the healthy and inexpensive meal preparation from discount store-bought food. Holding MedlinePlus training programs in conjunction with a cooking program and collaborating with local public libraries might be a promising format that needs additional research.

The long road of drug development for endometriosis - Pains, gains, and hopes.

Endometriosis, defined by the growth of endometrial tissues outside of the uterine cavity, is a global health burden for ∼200 million women. Patients with endometriosis usually present with chronic pain and are often diagnosed with infertility. The pathogenesis of endometriosis is still an open question; however, tissue stemness and immunological and genetic factors have been extensively discussed in the establishment of endometriotic lesions. Current treatments for endometriosis can be categorized into pharmacological management of hormone levels and surgical removal of the lesions. Both approaches have limited efficacy, with recurrences often encountered; thus, there is no complete cure for the disease or its symptoms. We review the current knowledge of the etiology of endometriosis and summarize the advancement of pharmacological management of endometriosis. We also discuss our efforts in applying DNA-encoded chemistry technology (DEC-Tec) to identify bioactive molecules for the treatment of endometriosis, offering new avenues for developing non-hormonal treatment options for those patients who seek spontaneous pregnancies.

The Mycobiota of Library Books in Russia.

Abstract-Numerous studies of microorganisms isolated from the surface of cultural heritage objects, including library documents, are regularly carried out in different countries. Although the micromycete composition in each case varies, some species are constantly isolated. The structure of micromycete communities that inhabit library documents was studied in 57 cities of Russia located in seven federal districts (Northwestern, Central, Southern, Volga, Ural, Siberian, and Far Eastern). Micromycetes of 95 species from 32 genera were isolated and identified. The mycobiota of the library documents represented by Ascomycota has more than 90% of the species richness; Mucoromycota make up 3-9% and Basidiomycota, 3-4%. The Aspergillaceae family was the leading family: it accounted for 48.5-67.3% of the total species richness. In all regions, species diversity is moderate: the Shannon index ranged from 2.7 to 3.3. The Mcintosh species richness index is high everywhere (48-126), except in the Ural district (15.3). The McIntosh (0.76-0.84) and Pielow (0.80-0.91) dominance indices indicate a high level of species evenness in the mycobiota. The obtained values demonstrate the stability the document mycobiota in libraries from different regions. Significant species similarity between the districts was revealed by calculation of binary coefficients: the Jaccard coefficient was from 0.44 to 0.60; the Sørensen's qualitative measure of similarity was from 0.63 to 0.75; the quantitative similarity measure of Sørensen was from 0.44 to 0.71, and Morisita-Horn was from 0.66 to 1.0. The groups of dominant species in different regions are quite similar. The study of the ecological diversity of mycobiota on library books demonstrated the moderate diversity and the stability of the community. A high degree of similarity of taxonomic structures was established regardless of the climate conditions of the regions. Cosmopolitans characterized by high frequency of occurrence formed the major core of the library book mycobiota: Alternaria alternata, Aspergillus niger, Cladosporium cladosporioides, Mucor plumbeus, and Penicillium aurantiogriseum.

Enzymatic Cleavage of Double-Stranded DNA-Encoded Libraries (DELs) to Single-Stranded DELs with Compounds at the 3' End: Its Application in Photo-crosslinking Selection.

DNA-encoded library (DEL) technology is a crucial tool in pharmaceutical research, rapidly identifying compounds that bind to a target of interest from an extensive pool of compounds. In this study, we propose a new method for generating single-stranded DELs (ssDELs) with compounds at the 3' end. The introduction of uniquely designed hairpin-shaped headpieces containing deoxyuridine (NC-HP) and the use of a cleavage enzyme facilitate the conversion from double-stranded DELs (dsDELs) to such ssDELs. Moreover, Klenow fill-in provides the dsDELs with photo-crosslinkers covalently linked to the coding region, which exhibit durability even under stringent washing conditions and enable photo-crosslinking with a high signal-to-noise ratio, as also confirmed in cell-based photo-crosslinking selections.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.