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The cooperative and participatory development of concepts, processes and spaces is becoming increasingly important for libraries. For library teams as well as for administrations it is vital that participatory processes can be initiated in easy ways regarding time and money. The article addresses this question and shows how the use of simulation games can lead to constructive solutions. Furthermore, it presents the "hochdrei – vision game" provided by the German Federal Cultural Foundation – a digital tool to design a simulation game specifically for libraries. [ABSTRACT FROM AUTHOR]

The unique palindromic inverted terminal repeats (ITRs) and single-stranded nature of adeno-associated virus (AAV) DNA are major hurdles to current sequencing technologies. Due to these characteristics, sequencing noncanonical AAV genomes present in AAV vector preparations remains challenging. To address this limitation, we developed thorough molecule configuration analysis of noncanonical AAV genomes (TMCA-AAV-seq). TMCA-AAV-seq takes advantage of the documented AAV packaging mechanism in which encapsidation initiates from its 3′ ITR, for AAV-seq library construction. Any AAV genome with a 3′ ITR is converted to a template suitable to adapter addition by a Bst DNA polymerase-mediated extension reaction. This extension reaction helps fix ITR heterogeneity in the AAV population and allows efficient adapter addition to even noncanonical AAV genomes. The resulting library maintains the original AAV genome configurations without introducing undesired changes. Subsequently, long-read sequencing can be performed by the Pacific Biosciences (PacBio) single-molecule, real-time (SMRT) sequencing technology platform. Finally, through comprehensive data analysis, we can recover canonical, noncanonical AAV DNA, and non-AAV vector DNA sequences, along with their molecular configurations. Our method is a robust tool for profiling thorough AAV-population genomes. TMCA-AAVseq can be further extended to all parvoviruses and their derivative vectors.

The emergence of antibiotic-resistant-bacteria is a serious public health threat, which prompts us to speed up the discovery of novel antibacterial agents. Phage display technology has great potential to screen peptides or antibodies with high binding capacities for a wide range of targets. This property is significant in the rapid search for new antibacterial agents for the control of bacterial resistance. In this paper, we not only summarized the recent progress of phage display for the discovery of novel therapeutic agents, identification of action sites of bacterial target proteins, and rapid detection of different pathogens, but also discussed several problems of this technology that must be solved. Breakthrough in these problems may further promote the development and application of phage display technology in the biomedical field in the future. [ABSTRACT FROM AUTHOR]

Objective: Solid-phase reversible immobilization (SPRI) beads are widely used for high-throughput sequencing library construction to purify and recover nucleic acids. This research was aimed at investigating the effects of SPRI bead ratio, incubation time, and elution time on nucleic acid recovery during fulllength 16S rDNA high-throughput sequencing library construction. Methods: The effects of different SPRI bead ratios, incubation times, and elution times were compared for three different initial sample amounts. An L9(3³) orthogonal experiment was designed to determine the optimal combination of these factors. Results: The incubation time of three factors including SPRI beads ratio, incubation time, and elution time had a statistically significant effect on the recovery rate for the initial sample amount of 1500 ng and 3000 ng. The orthogonal experiment results indicated that incubation time had the greatest impact among the three factors. Conclusion: Incubation time significantly influences recovery rate in full-length 16S rDNA high-throughput sequencing library construction. The use of 0.8× SPRI beads, 15 minutes of incubation, and 10 minutes of elution resulted in the highest recovery rate. SPRI beads offer a viable method for recovering fulllength 16S rDNA amplicons. [ABSTRACT FROM AUTHOR]

Affinity maturation is often a necessary step for the development of potent therapeutic molecules. Many different diversification strategies have been used for antibody affinity maturation, including error-prone PCR, chain shuffling, and targeted complementary-determining region (CDR) mutation. Although effective, they can negatively impact antibody stability or alter epitope recognition. Moreover, they do not address the presence of sequence liabilities, such as glycosylation, asparagine deamidation, aspartate isomerization, aggregation motifs, and others. Such liabilities, if present or inadvertently introduced, can potentially create the need for new rounds of engineering, or even abolish the value of the antibody as a therapeutic molecule. Here, we demonstrate a sequence agnostic method to improve antibody affinities, while simultaneously eliminating sequence liabilities and retaining the same epitope binding as the parental antibody. This was carried out using a defined collection of natural CDRs as the diversity source, purged of sequence liabilities, and matched to the antibody germline gene family. These CDRs were inserted into the lead molecule in one or two sites at a time (LCDR1-2, LCDR3, HCDR1-2) while retaining the HCDR3 and framework regions unchanged. The final analysis of 92 clones revealed 81 unique variants, with each of 24 tested variants having the same epitope specificity as the parental molecule. Of these, the average affinity improved by over 100 times (to 96 pM), and the best affinity improvement was 231-fold (to 32 pM).Abbreviations CDR: complementarity determining region; FACS: fluorescence-activated cell sorting; ka: association rate; kd: dissociation rate; KD: dissociation constant; scFv: single-chain variable fragment; SPR: surface plasmon resonance

Phage display is a well-established technology for in vitro selection of monoclonal antibodies (mAb), and more than 12 antibodies isolated from phage displayed libraries of different formats have been approved for therapy. We have constructed a large size (10^11) human antibody VH domain library based on thermo-stable, aggregation-resistant scaffolds. This diversity was obtained by grafting naturally occurring CDR2s and CDR3s from healthy donors with optimized primers into the VH library. This phage-displayed library was used for bio-panning against various antigens. So far, panels of binders have been isolated against different viral and tumor targets, including the SARS-CoV-2 RBD, HIV-1 ENV protein, mesothelin and FLT3. In the present study, we discuss domain library construction, characterize novel VH binders against human CD22 and PD-L1, and define our design process for antibody domain drug conjugation (DDC) as tumoricidal reagents. Our study provides examples for the potential applications of antibody domains derived from library screens in therapeutics and provides key information for large size human antibody domain library construction. [ABSTRACT FROM AUTHOR]

Phage display is a well-established technology for in vitro selection of monoclonal antibodies (mAb), and more than 12 antibodies isolated from phage displayed libraries of different formats have been approved for therapy. We have constructed a large size (10^11) human antibody VH domain library based on thermo-stable, aggregation-resistant scaffolds. This diversity was obtained by grafting naturally occurring CDR2s and CDR3s from healthy donors with optimized primers into the VH library. This phage-displayed library was used for bio-panning against various antigens. So far, panels of binders have been isolated against different viral and tumor targets, including the SARS-CoV-2 RBD, HIV-1 ENV protein, mesothelin and FLT3. In the present study, we discuss domain library construction, characterize novel VH binders against human CD22 and PD-L1, and define our design process for antibody domain drug conjugation (DDC) as tumoricidal reagents. Our study provides examples for the potential applications of antibody domains derived from library screens in therapeutics and provides key information for large size human antibody domain library construction.

The aim of the current article is to present a comprehensive and systematic review of the history of the National Southwest Associated University Library. The focus is on the construction process of the Library during 1937-1946. Toughened by working under the war-ridden and beleaguered conditions, and confronted with such challenges as tightening budgets and excessive scarcity of library facilities (books and reference materials, equipment and instruments) as well as personnel and supplies, library colleagues of Southwest Associated University endured great hardships in guarding book resources and disseminating knowledge together with other colleagues. They were all committed to preserving in library development, standardizing the library workflows, replenishing literature resources and developing various kinds of professional activities in libraries, which provided comprehensive library services for the teaching and scientific research of Southwest Associated University. Their spiritual outlooks and farsightedness are truly admirable. (Article content in Chinese with English extended abstract)

We have developed a rapid, quantitative method for analysing the outcome of the first strand synthesis step in cDNA library preparation, yield and molecular weight range of the final cDNA products are determined after size fractionation. This method involves conventional cDNA library construction including all enzymatic steps usually required, but replaces radioactive labelling of nucleic acids with fluorescence detection. The separation and quantification steps all involve ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC). This quantitative method replaces the use of autoradiography and size exclusion chromatography with combined ion-pair reversed-phase high performance liquid chromatography and in line fluorescence detection. The result of this approach is combination of speed with the generation of reproducible, high quality cDNA libraries.

The emergence of antibiotic-resistant-bacteria is a serious public health threat, which prompts us to speed up the discovery of novel antibacterial agents. Phage display technology has great potential to screen peptides or antibodies with high binding capacities for a wide range of targets. This property is significant in the rapid search for new antibacterial agents for the control of bacterial resistance. In this paper, we not only summarized the recent progress of phage display for the discovery of novel therapeutic agents, identification of action sites of bacterial target proteins, and rapid detection of different pathogens, but also discussed several problems of this technology that must be solved. Breakthrough in these problems may further promote the development and application of phage display technology in the biomedical field in the future.

Objective To build a construction strategy for RNA-seq library for small quantity of cells by optimizing Tn5 transposase assembly and purification workflow. Methods Recombinant plasmid pSUMO-Tn5 was constructed by homologous recombination. After Tn5 protein was induced to express, the product was purified with affinity chromatography followed by optimization of Tn5 assembly. Then we constructed RNA-seq library using RAW264.7 cells and analyzed the number of genes detected and the ratio of unmapped reads. Results Soluble Tn5 protein was expressed in Codon Plus BL21 with the recombinant plasmid pSUMO-Tn5. Tn5 with high purity was obtained through affinity purification. RNA-seq library for small quantity of RAW264.7 cells was successfully constructed. After the optimization of Tn5 assembly, nonspecific amplification was remarkably reduced compared with the control (P < 0.05). Conclusion Tn5 protein is effectively expressed and purified. Ultra-filtered Tn5 transposase can reduce the nonspecific amplification in RNA-seq, providing a new strategy of RNA-seq for small quantity of cells.

The analysis of cell-free circulating tumor DNA (ctDNA) is potentially a less invasive, more dynamic assessment of cancer progression and treatment response than characterizing solid tumor biopsies. Standard isolation methods require separation of plasma by centrifugation, a time-consuming step that complicates automation. To address these limitations, we present an automatable magnetic bead-based ctDNA isolation method that eliminates centrifugation to purify ctDNA directly from peripheral blood (PB). To develop and test our method, ctDNA from cancer patients was purified from PB and plasma. We found that allelic fractions of somatic single-nucleotide variants from target gene capture libraries were comparable, indicating that the PB ctDNA purification method may be a suitable replacement for the plasma-based protocols currently in use.

Background: Validation and standardization of methodologies for microbial community measurements by high-throughput sequencing are needed to support human microbiome research and its industrialization. This study set out to establish standards-based solutions to improve the accuracy and reproducibility of metagenomics-based microbiome profiling of human fecal samples. Results: In the first phase, we performed a head-to-head comparison of a wide range of protocols for DNA extraction and sequencing library construction using defined mock communities, to identify performant protocols and pinpoint sources of inaccuracy in quantification. In the second phase, we validated performant protocols with respect to their variability of measurement results within a single laboratory (that is, intermediate precision) as well as interlaboratory transferability and reproducibility through an industry-based collaborative study. We further ascertained the performance of our recommended protocols in the context of a community-wide interlaboratory study (that is, the MOSAIC Standards Challenge). Finally, we defined performance metrics to provide best practice guidance for improving measurement consistency across methods and laboratories. Conclusions: The validated protocols and methodological guidance for DNA extraction and library construction provided in this study expand current best practices for metagenomic analyses of human fecal microbiota. Uptake of our protocols and guidelines will improve the accuracy and comparability of metagenomics-based studies of the human microbiome, thereby facilitating development and commercialization of human microbiome-based products. 14cWN4qpGoEHsF7NJvL5FB Video Abstract [ABSTRACT FROM AUTHOR]

Background Cholesterol gallstone (CG) is the most common gallstone disease, which is induced by biliary cholesterol supersaturation. The purpose of this study is to investigate the pathogenesis of CG. Methods Sixteen mice were equally and randomly divided into model group and normal control group. The model group was fed with lithogenic diets to induce CG, and then gallbladder bile lipid analysis was performed. After RNA-seq library was constructed, differentially expressed mRNAs (DE-mRNAs) and differentially expressed lncRNAs (DE-lncRNAs) between model group and normal control group were analyzed by DESeq2 package. Using the cluster Profiler package, enrichment analysis for the DE-mRNAs was carried out. Based on Cytoscape software, the protein-protein interaction (PPI) network and competing endogenous RNA (ceRNA) network were built. Using quantitative real-time reverse transcription-PCR (qRT-PCR) analysis, the key RNAs were validated. Results The mouse model of CG was suc cessfully established, and then 181 DE-mRNAs and 33 DE-lncRNAs between model and normal groups were obtained. Moreover, KDM4A was selected as a hub node in the PPI network, and lncRNA MEG3 was considered as a key lncRNA in the regulatory network. Additionally, the miR-107-5p/miR-149-3p/miR-346-3-MEG3 regulatory pairs and MEG3-PABPC4/CEP131/NUMB1 co-expression pairs existed in the regulatory network. The qRT-PCR analysis showed that KDM4A expression was increased, and the expressions of MEG3, PABPC4, CEP131, and NUMB1 were downregulated. Conclusion These RNAs might be related to the pathogenesis of CG.

Abstract Background Next-generation sequencing (NGS) is fundamental to the current biological and biomedical research. Construction of sequencing library is a key step of NGS. Therefore, various library construction methods have been explored. However, the current methods are still limited by some shortcomings. Results This study developed a new NGS library construction method, Single strand Adaptor Library Preparation (SALP), by using a novel single strand adaptor (SSA). SSA is a double-stranded oligonucleotide with a 3′ overhang of 3 random nucleotides, which can be efficiently ligated to the 3′ end of single strand DNA by T4 DNA ligase. SALP can be started with any denatured DNA fragments such as those sheared by Tn5 tagmentation, enzyme digestion and sonication. When started with Tn5-tagmented chromatin, SALP can overcome a key limitation of ATAC-seq and become a high-throughput NGS library construction method, SALP-seq, which can be used to comparatively characterize the chromatin openness state of multiple cells unbiasly. In this way, this study successfully characterized the comparative chromatin openness states of four different cell lines, including GM12878, HepG2, HeLa and 293T, with SALP-seq. Similarly, this study also successfully characterized the chromatin openness states of HepG2 cells with SALP-seq by using 105 to 500 cells. Conclusions This study developed a new NGS library construction method, SALP, by using a novel kind of single strand adaptor (SSA), which should has wide applications in the future due to its unique performance.

The aim of the current article is to present a comprehensive and systematic review of the history of the National Southwest Associated University Library. The focus is on the construction process of the Library during 1937-1946. Toughened by working under the war-ridden and beleaguered conditions, and confronted with such challenges as tightening budgets and excessive scarcity of library facilities （books and reference materials, equipment and instruments） as well as personnel and supplies, library colleagues of Southwest Associated University endured great hardships in guarding book resources and disseminating knowledge together with other colleagues. They were all committed to preserving in library development, standardizing the library workflows, replenishing literature resources and developing various kinds of professional activities in libraries, which provided comprehensive library services for the teaching and scientific research of Southwest Associated University. Their spiritual outlooks and farsightedness are truly admirable. [ABSTRACT FROM AUTHOR]

Background: Candida auris is characterized for having a high genetic variability among species. MALDI-TOF MS library contains spectra from only three strains of C. auris, which makes difficult the identification process and gives low scores at the species level. Our aim was to construct and validate an internal library to improve C. auris identification with Colombian clinical strains. Methods: From 30 clinical strains, 770 mass spectra were obtained for the construction of the database. The validation was performed with 300 strains to compare the identification results in the BDAL and C. auris Colombia libraries. Results: Our library allowed a complete, 100% identification of the evaluated strains and a significant improvement in the scores obtained, showing a better performance compared to the Bruker BDAL library. Conclusions: The strengthening of the database is a great opportunity to improve the scoring and C. auris identification. Library data are available via ProteomeXchange with identifier PXD016387. [ABSTRACT FROM AUTHOR]

Abstract Background RNA-Sequencing (RNA-seq) is now commonly used to reveal quantitative spatiotemporal snapshots of the transcriptome, the structures of transcripts (splice variants and fusions) and landscapes of expressed mutations. However, standard approaches for library construction typically require relatively high amounts of input RNA, are labor intensive, and are time consuming. Methods Here, we report the outcome of a systematic effort to optimize and streamline steps in strand-specific RNA-seq library construction. Results This work has resulted in the identification of an optimized messenger RNA isolation protocol, a potent reverse transcriptase for cDNA synthesis, and an efficient chemistry and a simplified formulation of library construction reagents. We also present an optimization of bead-based purification and size selection designed to maximize the recovery of cDNA fragments. Conclusions These developments have allowed us to assemble a rapid high throughput pipeline that produces high quality data from amounts of total RNA as low as 25 ng. While the focus of this study is on RNA-seq sample preparation, some of these developments are also relevant to other next-generation sequencing library types.

The unique palindromic inverted terminal repeats (ITRs) and single-stranded nature of adeno-associated virus (AAV) DNA are major hurdles to current sequencing technologies. Due to these characteristics, sequencing noncanonical AAV genomes present in AAV vector preparations remains challenging. To address this limitation, we developed thorough molecule configuration analysis of noncanonical AAV genomes (TMCA-AAV-seq). TMCA-AAV-seq takes advantage of the documented AAV packaging mechanism in which encapsidation initiates from its 3' ITR, for AAV-seq library construction. Any AAV genome with a 3' ITR is converted to a template suitable to adapter addition by a Bst DNA polymerase-mediated extension reaction. This extension reaction helps fix ITR heterogeneity in the AAV population and allows efficient adapter addition to even noncanonical AAV genomes. The resulting library maintains the original AAV genome configurations without introducing undesired changes. Subsequently, long-read sequencing can be performed by the Pacific Biosciences (PacBio) single-molecule, real-time (SMRT) sequencing technology platform. Finally, through comprehensive data analysis, we can recover canonical, noncanonical AAV DNA, and non-AAV vector DNA sequences, along with their molecular configurations. Our method is a robust tool for profiling thorough AAV-population genomes. TMCA-AAVseq can be further extended to all parvoviruses and their derivative vectors., Competing Interests: W.X. holds equity in Ivygen Corporation and Nikegen., (© 2024 The Authors.)

The maize mutant gene Vestigial glume 1 (Vg1) has been fine-mapped to a narrow region by map-based cloning and the candidate gene for Vg1 spanned 19.5 kb. Here we report Vg1 genomic fosmid library construction and screening. The fosmid library of Vg1 consisted of 574,000 clones with an average insert size of 36.4 kb, representing 7.9-fold coverage of the maize genome. Fosmid stability assays indicated that clones were stable during propagation in the fosmid system. Using Vg1 candidate gene-specific primers, a positive clone was successfully identified. This discovery will pave the way for identifying the function of Vg1 in maize development.

Background: Candida auris is characterized for having a high genetic variability among species. MALDI-TOF MS library contains spectra from only three strains of C. auris, which makes difficult the identification process and gives low scores at the species level. Our aim was to construct and validate an internal library to improve C. auris identification with Colombian clinical strains. Methods: From 30 clinical strains, 770 mass spectra were obtained for the construction of the database. The validation was performed with 300 strains to compare the identification results in the BDAL and C. auris Colombia libraries. Results: Our library allowed a complete, 100% identification of the evaluated strains and a significant improvement in the scores obtained, showing a better performance compared to the Bruker BDAL library. Conclusions: The strengthening of the database is a great opportunity to improve the scoring and C. auris identification. Library data are available via ProteomeXchange with identifier PXD016387.

Metagenomic techniques, notably the cloning of environmental DNA (eDNA) into surrogate hosts, have given access to the genome of uncultured bacteria. However, the determination of gene functions based on DNA sequences alone remains a significant challenge. The functional screening of metagenomic libraries represents an interesting approach in the discovery of microbial metabolites. We describe here an optimized screening approach that facilitates the identification of new antimicrobials among large metagenomic libraries. Notably, we report a detailed genomic library construction protocol using Escherichia coli DH10B as a surrogate host, and demonstrate how vector/genomic DNA dephosphorylation, ligase inactivation, dialysis of the ligation product and vector/genomic DNA ratio greatly influence clone recovery. Furthermore, we describe the use of an airbrush device to screen E. coli metagenomic libraries for their antibacterial activity against Staphylococcus aureus, a method we called bacteriospray. This bacterial spraying tool greatly facilitates and improves the functional screening of large genomic libraries, as it conveniently allows the production of a thinner and more uniform layer of target bacteria compared to the commonly used overlay method, resulting in the screening of 5–10 times more clones per agar plate. Using the Burkholderia thailandensis E264 genomic DNA as a proof of concept, four clones out of 70,000 inhibited the growth of S. aureus and were found to each contain a DNA insert. Analysis of these chromosomic fragments revealed genomic regions never previously reported to be responsible for the production of antimicrobials, nor predicted by bioinformatics tools.

A transposon-based approach for the construction of sequencing libraries is an efficient way of preparing samples for processing on both Illumina and Ion Torrent platforms. However, PCR-mediated incorporation of adaptors in tagged DNA fragments leaves behind self-complementary regions flanking the DNA fragment. These regions are capable of forming hairpin structures and, together with adaptors, create conditions for the potential formation of template heteroduplexes. These negatively affect the sequencing process on the Ion Torrent platform and can lead to a more than 3-fold decline in output data compared with sequencing of conventional libraries. To address this problem, we have developed MuPlus, a transposon-based protocol for barcoded library preparation for Ion Torrent, in which one adaptor is integrated by PCR and the second is integrated by ligation as a single-stranded oligonucleotide after enzymatic cleavage of a complementary part on one strand of the tag. The resulting library does not contain self-complementary, hairpin-forming regions, is free of heteroduplexes, and can be analyzed on the Ion Torrent platform with the same efficiency as a library created with a ligation-based protocol.

Site-saturation mutagenesis is a powerful tool for protein optimization due to its efficiency and simplicity. A degenerate codon NNN or NNS (K) is often used to encode the 20 standard amino acids, but this will produce redundant codons and cause uneven distribution of amino acids in the constructed library. Here we present a novel “small-intelligent” strategy to construct mutagenesis libraries that have a minimal gene library size without inherent amino acid biases, stop codons, or rare codons of Escherichia coli by coupling well-designed combinatorial degenerate primers with suitable PCR-based mutagenesis methods. The designed primer mixture contains exactly one codon per amino acid and thus allows the construction of small-intelligent mutagenesis libraries with one gene per protein. In addition, the software tool DC-Analyzer was developed to assist in primer design according to the user-defined randomization scheme for library construction. This small-intelligent strategy was successfully applied to the randomization of halohydrin dehalogenases with one or two randomized sites. With the help of DC-Analyzer, the strategy was proven to be as simple as NNS randomization and could serve as a general tool to efficiently randomize target genes at positions of interest.

Three new lipolytic genes were isolated from a forest soil metagenomic library by functional screening on tributyrin agar plates. The genes SBLip1, SBLip2 and SBLip5.1 respectively encode polypeptides of 445, 346 and 316 amino acids. Phylogenetic analyses revealed that SBLip2 and SBLip5.1 belong to bacterial esterase/lipase family IV, whereas SBLip1 shows similarity to class C β-lactamases and is thus related to esterase family VIII. The corresponding genes were overexpressed and their products purified by affinity chromatography for characterization. Analyses of substrate specificity with different p-nitrophenyl esters showed that all three enzymes have a preference for short-acyl-chain p-nitrophenyl esters, a feature of carboxylesterases as opposed to lipases. The β-lactamase activity of SBLip1, measured with the chromogenic substrate nitrocefin, was very low. The three esterases have the same optimal pH (pH 10) and remain active across a relatively broad pH range, displaying more than 60 % activity between pH 6 and 10. The temperature optima determined were 35 °C for SBLip1, 45 °C for SBLip2 and 50 °C for SBLip5.1. The three esterases displayed different levels of tolerance to salts, solvents and detergents, SBLip2 being overall more tolerant to high concentrations of solvent and SBLip5.1 less affected by detergents. [ABSTRACT FROM AUTHOR]

In the last two decades, several phage display-selected monoclonal antibodies (mAbs) have been described in the literature and a few of them have managed to reach the clinics. Among these, the anti-respiratory syncytial virus (RSV) Palivizumab, a phage-display optimized mAb, is the only marketed mAb directed against microbial pathogens. Palivizumab is a clear example of the importance of choosing the most appropriate strategy when selecting or optimizing an anti-infectious mAb. From this perspective, the extreme versatility of phage-display technology makes it a useful tool when setting up different strategies for the selection of mAbs directed against human pathogens, especially when their possible clinical use is considered. In this paper, we review the principal phage display strategies used to select anti-infectious mAbs, with particular attention focused on those used against hypervariable pathogens, such as HCV and influenza viruses [ABSTRACT FROM AUTHOR]

Cardiac fibrosis (CF) and heart failure (HF) are common heart diseases, and severe CF can lead to HF. In this study, we tried to find their common potential molecular markers, which may help the diagnosis and treatment of CF and HF. RNA library construction and high-throughput sequencing were performed. The DESeq2 package in R was used to screen differentially expressed mRNAs (DEmRNAs), differentially expressed lncRNA (DElncRNAs) and differentially expressed miRNA (DEmiRNAs) between different samples. The common DEmRNAs, DElncRNAs and DEmiRNAs for the two diseases were obtained. The ConsensusPathDB (CPDB) was used to perform biological function enrichment for common DEmRNAs. Gene interaction network was constructed to screen out key genes. Subsequently, real-time polymerase chain reaction (RT-PCR) verification was performed. Lastly, GSE104150 and GSE21125 data sets were utilized for expression validation and diagnostic analysis. There were 1477 DEmRNAs, 502 DElncRNAs and 36 DEmiRNAs between CF and healthy control group. There were 607 DEmRNAs, 379DElncRNAs,s and 42 DEmiRNAs between HF and healthy control group. CH and FH shared 146 DEmRNAs, 80 DElncRNAs, and 6 DEmiRNAs. Hsa-miR-144-3p, CCNE2, C9orf72, MAP3K20-AS1, LEF1-AS1, AC243772.2, FLJ46284, and AC239798.2 were key molecules in lncRNA-miRNA-mRNA network. In addition, hsa-miR-144-3p and CCNE2 may be considered as potential diagnostic gene biomarkers in HF. In this study, the identification of common biomarkers of CF and HF may help prevent CF to HF transition as early as possible.

Abbreviations: CDR: complementarity determining region; FACS: fluorescence-activated cell sorting; k a : association rate; k d : dissociation rate; K D : dissociation constant; scFv: single-chain variable fragment; SPR: surface plasmon resonance.

Microbes employ sophisticated cellular networks encoded by complex genomes to rapidly adapt to changing environments. High-throughput genome engineering methods are valuable tools for functionally profiling genotype-phenotype relationships and understanding the complexity of cellular networks. However, current methods either rely on special homologous recombination systems and are thus applicable in only limited bacterial species or can generate only nonspecific mutations and thus require extensive subsequent screening. Here, we report a site-specific transposon-assisted genome engineering (STAGE) method that allows high-throughput Cas12k-guided mutagenesis in various microorganisms, such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Exploiting the powerful STAGE technique, we construct a site-specific transposon mutant library that focuses on all possible transcription factors (TFs) in P. aeruginosa, enabling the comprehensive identification of essential genes and antibiotic-resistance-related factors. Given its broad host range activity and easy programmability, this method can be widely adapted to diverse microbial species for rapid genome engineering and strain evolution., Competing Interests: Declaration of interests A patent application has been submitted for the Cas12k-guided transposition system., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Cholesterol gallstone (CG) is the most common gallstone disease, which is induced by biliary cholesterol supersaturation. The purpose of this study is to investigate the pathogenesis of CG., Methods: Sixteen mice were equally and randomly divided into model group and normal control group. The model group was fed with lithogenic diets to induce CG, and then gallbladder bile lipid analysis was performed. After RNA-seq library was constructed, differentially expressed mRNAs (DE-mRNAs) and differentially expressed lncRNAs (DE-lncRNAs) between model group and normal control group were analyzed by DESeq2 package. Using the cluster Profiler package, enrichment analysis for the DE-mRNAs was carried out. Based on Cytoscape software, the protein-protein interaction (PPI) network and competing endogenous RNA (ceRNA) network were built. Using quantitative real-time reverse transcription-PCR (qRT-PCR) analysis, the key RNAs were validated., Results: The mouse model of CG was suc cessfully established, and then 181 DE-mRNAs and 33 DE-lncRNAs between model and normal groups were obtained. Moreover, KDM4A was selected as a hub node in the PPI network, and lncRNA MEG3 was considered as a key lncRNA in the regulatory network. Additionally, the miR-107-5p/miR-149-3p/miR-346-3-MEG3 regulatory pairs and MEG3-PABPC4/CEP131/NUMB1 co-expression pairs existed in the regulatory network. The qRT-PCR analysis showed that KDM4A expression was increased, and the expressions of MEG3 , PABPC4 , CEP131 , and NUMB1 were downregulated., Conclusion: These RNAs might be related to the pathogenesis of CG., Competing Interests: The authors declare there are no competing interests., (©2021 Qian et al.)

The analysis of cell-free circulating tumor DNA (ctDNA) is potentially a less invasive, more dynamic assessment of cancer progression and treatment response than characterizing solid tumor biopsies. Standard isolation methods require separation of plasma by centrifugation, a time-consuming step that complicates automation. To address these limitations, we present an automatable magnetic bead-based ctDNA isolation method that eliminates centrifugation to purify ctDNA directly from peripheral blood (PB). To develop and test our method, ctDNA from cancer patients was purified from PB and plasma. We found that allelic fractions of somatic single-nucleotide variants from target gene capture libraries were comparable, indicating that the PB ctDNA purification method may be a suitable replacement for the plasma-based protocols currently in use.

Metagenomic techniques, notably the cloning of environmental DNA (eDNA) into surrogate hosts, have given access to the genome of uncultured bacteria. However, the determination of gene functions based on DNA sequences alone remains a significant challenge. The functional screening of metagenomic libraries represents an interesting approach in the discovery of microbial metabolites. We describe here an optimized screening approach that facilitates the identification of new antimicrobials among large metagenomic libraries. Notably, we report a detailed genomic library construction protocol using Escherichia coli DH10B as a surrogate host, and demonstrate how vector/genomic DNA dephosphorylation, ligase inactivation, dialysis of the ligation product and vector/genomic DNA ratio greatly influence clone recovery. Furthermore, we describe the use of an airbrush device to screen E. coli metagenomic libraries for their antibacterial activity against Staphylococcus aureus , a method we called bacteriospray. This bacterial spraying tool greatly facilitates and improves the functional screening of large genomic libraries, as it conveniently allows the production of a thinner and more uniform layer of target bacteria compared to the commonly used overlay method, resulting in the screening of 5-10 times more clones per agar plate. Using the Burkholderia thailandensis E264 genomic DNA as a proof of concept, four clones out of 70,000 inhibited the growth of S. aureus and were found to each contain a DNA insert. Analysis of these chromosomic fragments revealed genomic regions never previously reported to be responsible for the production of antimicrobials, nor predicted by bioinformatics tools.