Construction of transcript regulation mechanism prediction models based on binding motif environment of transcription factor AoXlnR in Aspergillus oryzae.

DNA-binding transcription factors (TFs) play a central role in transcriptional regulation mechanisms, mainly through their specific binding to target sites on the genome and regulation of the expression of downstream genes. Therefore, a comprehensive analysis of the function of these TFs will lead to the understanding of various biological mechanisms. However, the functions of TFs in vivo are diverse and complicated, and the identified binding sites on the genome are not necessarily involved in the regulation of downstream gene expression. In this study, we investigated whether DNA structural information around the binding site of TFs can be used to predict the involvement of the binding site in the regulation of the expression of genes located downstream of the binding site. Specifically, we calculated the structural parameters based on the DNA shape around the DNA binding motif located upstream of the gene whose expression is directly regulated by one TF AoXlnR from Aspergillus oryzae, and showed that the presence or absence of expression regulation can be predicted from the sequence information with high accuracy ([Formula: see text]-1.0) by machine learning incorporating these parameters.

FcRY is a key molecule controlling maternal blood IgY transfer to yolks during egg development in avian species.

Maternal immunoglobulin transfer plays a key role in conferring passive immunity to neonates. Maternal blood immunoglobulin Y (IgY) in avian species is transported to newly-hatched chicks in two steps: 1) IgY is transported from the maternal circulation to the yolk of maturing oocytes, 2) the IgY deposited in yolk is transported to the circulation of the embryo via the yolk sac membrane. An IgY-Fc receptor, FcRY, is involved in the second step, but the mechanism of the first step is still unclear. We determined whether FcRY was also the basis for maternal blood IgY transfer to the yolk in the first step during egg development. Immunohistochemistry revealed that FcRY was expressed in the capillary endothelial cells in the internal theca layer of the ovarian follicle. Substitution of the amino acid residue in Fc region of IgY substantially changed the transport efficiency of IgY into egg yolks when intravenously-injected into laying quail; the G365A mutant had a high transport efficiency, but the Y363A mutant lacked transport ability. Binding analyses of IgY mutants to FcRY indicated that the mutant with a high transport efficiency (G365A) had a strong binding activity to FcRY; the mutants with a low transport efficiency (G365D, N408A) had a weak binding activity to FcRY. One exception, the Y363A mutant had a remarkably strong binding affinity to FcRY, with a small dissociation rate. The injection of neutralizing FcRY antibodies in laying quail markedly reduced IgY uptake into egg yolks. The neutralization also showed that FcRY was engaged in prolongation of half-life of IgY in the blood; FcRY is therefore a multifunctional receptor that controls avian immunity. The pattern of the transport of the IgY mutants from the maternal blood to the egg yolk was found to be identical to that from the fertilized egg yolk to the newly-hatched chick blood circulation, via the yolk sac membrane. FcRY is therefore a critical IgY receptor that regulates the IgY uptake from the maternal blood circulation into the yolk of avian species, further indicating that the two steps of maternal-newly-hatched IgY transfer are controlled by a single receptor.

The Identification of a Target Gene of the Transcription Factor KojR and Elucidation of Its Role in Carbon Metabolism for Kojic Acid Biosynthesis in Aspergillus oryzae.

DNA-binding transcription factors are broadly characterized as proteins that bind to specific sequences within genomic DNA and modulate the expression of downstream genes. This study focused on KojR, a transcription factor involved in the metabolism of kojic acid, which is an organic acid synthesized in Aspergillus oryzae and is known for its tyrosinase-inhibitory properties. However, the regulatory mechanism underlying KojR-mediated kojic acid synthesis remains unclear. Hence, we aimed to obtain a comprehensive identification of KojR-associated genes using genomic systematic evolution of ligands by exponential enrichment with high-throughput DNA sequencing (gSELEX-Seq) and RNA-Seq. During the genome-wide exploration of KojR-binding sites via gSELEX-Seq and identification of KojR-dependent differentially expressed genes (DEGs) using RNA-Seq, we confirmed that KojR preferentially binds to 5'-CGGCTAATGCGG-3', and KojR directly regulates kojT, as was previously reported. We also observed that kojA expression, which may be controlled by KojR, was significantly reduced in a ΔkojR strain. Notably, no binding of KojR to the kojA promoter region was detected. Furthermore, certain KojR-dependent DEGs identified in the present study were associated with enzymes implicated in the carbon metabolic pathway of A. oryzae. This strongly indicates that KojR plays a central role in carbon metabolism in A. oryzae.

Rapid and cost-effective epitope mapping using PURE ribosome display coupled with next-generation sequencing and bioinformatics.

A novel, efficient and cost-effective approach for epitope identification of an antibody has been developed using a ribosome display platform. This platform, known as PURE ribosome display, utilizes an Escherichia coli-based reconstituted cell-free protein synthesis system (PURE system). It stabilizes the mRNA-ribosome-peptide complex via a ribosome-arrest peptide sequence. This system was complemented by next-generation sequencing (NGS) and an algorithm for analyzing binding epitopes. To showcase the effectiveness of this method, selection conditions were refined using the anti-PA tag monoclonal antibody with the PA tag peptide as a model. Subsequently, a random peptide library was constructed using 10 NNK triplet oligonucleotides via the PURE ribosome display. The resulting random peptide library-ribosome-mRNA complex was selected using a commercially available anti-HA (YPYDVPDYA) tag monoclonal antibody, followed by NGS and bioinformatic analysis. Our approach successfully identified the DVPDY sequence as an epitope within the hemagglutinin amino acid sequence, which was then experimentally validated. This platform provided a valuable tool for investigating continuous epitopes in antibodies.

AtMYB50 regulates root cell elongation by upregulating PECTIN METHYLESTERASE INHIBITOR 8 in Arabidopsis thaliana.

Plant root development involves multiple signal transduction pathways. Notably, phytohormones like auxin and cytokinin are well characterized for their molecular mechanisms of action. Reactive oxygen species (ROS) serve as crucial signaling molecules in controlling root development. The transcription factor, UPBEAT1 (UPB1) is responsible for maintaining ROS homeostasis at the root tip, influencing the transition from cell proliferation to differentiation. While UPB1 directly regulates peroxidase expression to control ROS homeostasis, it targets genes other than peroxidases, suggesting its involvement in root growth through non-ROS signals. Our investigation focused on the transcription factor MYB50, a direct target of UPB1, in Arabidopsis thaliana. By analyzing multiple fluorescent proteins and conducting RNA-seq and ChIP-seq, we unraveled a step in the MYB50 regulatory gene network. This analysis, in conjunction with the UPB1 regulatory network, demonstrated that MYB50 directly regulates the expression of PECTIN METHYLESTERASE INHIBITOR 8 (PMEI8). Overexpressing PMEI8, similar to the MYB50, resulted in reduced mature cell length. These findings establish MYB50 as a regulator of root growth within the UPB1 gene regulatory network. Our study presents a model involving transcriptional regulation by MYB50 in the UPB1 regulated root growth system and sheds light on cell elongation via pectin modification.

The draft genome sequence of the Japanese rhinoceros beetle Trypoxylus dichotomus septentrionalis towards an understanding of horn formation.

The Japanese rhinoceros beetle Trypoxylus dichotomus is a giant beetle with distinctive exaggerated horns present on the head and prothoracic regions of the male. T. dichotomus has been used as a research model in various fields such as evolutionary developmental biology, ecology, ethology, biomimetics, and drug discovery. In this study, de novo assembly of 615 Mb, representing 80% of the genome estimated by flow cytometry, was obtained using the 10 × Chromium platform. The scaffold N50 length of the genome assembly was 8.02 Mb, with repetitive elements predicted to comprise 49.5% of the assembly. In total, 23,987 protein-coding genes were predicted in the genome. In addition, de novo assembly of the mitochondrial genome yielded a contig of 20,217 bp. We also analyzed the transcriptome by generating 16 RNA-seq libraries from a variety of tissues of both sexes and developmental stages, which allowed us to identify 13 co-expressed gene modules. We focused on the genes related to horn formation and obtained new insights into the evolution of the gene repertoire and sexual dimorphism as exemplified by the sex-specific splicing pattern of the doublesex gene. This genomic information will be an excellent resource for further functional and evolutionary analyses, including the evolutionary origin and genetic regulation of beetle horns and the molecular mechanisms underlying sexual dimorphism.

Nascent MSKIK peptide cancels ribosomal stalling by arrest peptides in Escherichia coli.

The insertion of the DNA sequence encoding SKIK peptide adjacent to the M start codon of a difficult-to-express protein enhances protein production in Escherichia coli. In this report, we reveal that the increased production of the SKIK-tagged protein is not due to codon usage of the SKIK sequence. Furthermore, we found that insertion of SKIK or MSKIK just before the SecM arrest peptide (FSTPVWISQAQGIRAGP), which causes ribosomal stalling on mRNA, greatly increased the production of the protein containing the SecM arrest peptide in the E. coli-reconstituted cell-free protein synthesis system (PURE system). A similar translation enhancement phenomenon by MSKIK was observed for the CmlA leader peptide, a ribosome arrest peptide, whose arrest is induced by chloramphenicol. These results strongly suggest that the nascent MSKIK peptide prevents or releases ribosomal stalling immediately following its generation during the translation process, resulting in an increase of protein production.

Bioinformatics in bioscience and bioengineering: Recent advances, applications, and perspectives.

Recent advances have led to the emergence of highly comprehensive and analytical approaches, such as omics analysis and high-resolution, time-resolved bioimaging analysis. These technologies have made it possible to obtain vast data from a single measurement. Subsequently, large datasets have pioneered the data-driven approach, an alternative to the traditional hypothesis-testing system, for researchers. However, processing, interpreting, and elucidating enormous datasets is no longer possible without computation. Bioinformatics is a field that has developed over long periods, intending to understand biological phenomena using methods collected from information science and statistics, thus solving this proposed research challenge. This review presents the latest methodologies and applications in sequencing, imaging, and mass spectrometry that were developed using bioinformatics. We presented the features of individual techniques and outlines in each part, avoiding the use of complex algorithms and formulas to allow beginning researchers to understand an overview. In the section on sequencing, we focused on comparative genomic, transcriptomic, and bacterial microbiome analyses, which are frequently used as applications of next-generation sequencing. Bioinformatic methods for handling sequence data and case studies were described. In the section on imaging, we introduced the analytical methods and microscopy imaging informatics techniques used in animal cell biology and plant physiology. We introduce informatics technologies for maximizing the value of measured data, including predicting the structure of unknown molecules and untargeted analysis in the section on mass spectrometry. Finally, we discuss the future outlook of this field. We anticipate that this review will assist biologists in using bioinformatics more effectively.

Comprehensive analysis of transglutaminase substrate preference by cDNA display coupled with next-generation sequencing and bioinformatics.

cDNA display is an in vitro display technology based on a covalent linkage between a protein and its corresponding mRNA/cDNA, widely used for the selection of proteins and peptides from large libraries (1012) in a high throughput manner, based on their binding affinity. Here, we developed a platform using cDNA display and next-generation sequencing (NGS) for rapid and comprehensive substrate profiling of transglutaminase 2 (TG2), an enzyme crosslinking glutamine and lysine residues in proteins. After screening and selection of the control peptide library randomized at the reactive glutamine, a combinatorial library of displayed peptides randomized at positions - 1, + 1, + 2, and + 3 from the reactive glutamine was screened followed by NGS and bioinformatic analysis, which indicated a strong preference of TG2 towards peptides with glutamine at position - 1 (Gln-Gln motif), and isoleucine or valine at position + 3. The highly enriched peptides indeed contained the indicated sequence and showed a higher reactivity as TG2 substrates than the peptide previously selected by phage display, thus representing the novel candidate peptide probes for TG2 research. Furthermore, the obtained information on substrate profiling can be used to identify potential TG2 protein targets. This platform will be further used for the substrate profiling of other TG isozymes, as well as for the selection and evolution of larger biomolecules.

Auxin Distribution in Lateral Root Primordium Development Affects the Size and Lateral Root Diameter of Rice.

Lateral roots (LRs) occupy a large part of the root system and play a central role in plant water and nutrient uptake. Monocot plants, such as rice, produce two types of LRs: the S-type (short and thin) and the L-type (long, thick, and capable of further branching). Because of the ability to produce higher-order branches, the L-type LR formation contributes to efficient root system expansion. Auxin plays a major role in regulating the root system development, but its involvement in developing different types of LRs is largely unknown. Here, we show that auxin distribution is involved in regulating LR diameter. Dynamin-related protein (DRP) genes were isolated as causative genes of the mutants with increased L-type LR number and diameter than wild-type (WT). In the drp mutants, reduced endocytic activity was detected in rice protoplast and LRs with a decreased OsPIN1b-GFP endocytosis in the protoplast. Analysis of auxin distribution using auxin-responsive promoter DR5 revealed the upregulated auxin signaling in L-type LR primordia (LRP) of the WT and the mutants. The application of polar auxin transport inhibitors enhanced the effect of exogenous auxin to increase LR diameter with upregulated auxin signaling in the basal part of LRP. Inducible repression of auxin signaling in the mOsIAA3-GR system suppressed the increase in LR diameter after root tip excision, suggesting a positive role of auxin signaling in LR diameter increase. A positive regulator of LR diameter, OsWOX10, was auxin-inducible and upregulated in the drp mutants more than the WT, and revealed as a potential target of ARF transcriptional activator. Therefore, auxin signaling upregulation in LRP, especially at the basal part, induces OsWOX10 expression, increasing LR diameter.

Administration of Aspergillus oryzae suppresses DSS-induced colitis.

Aspergillus oryzae, a filamentous fungus, has long been used for the production of traditional Japanese foods. Here, we analyzed how A. oryzae administration affects the intestinal environment in mice. The results of 16S rRNA gene sequencing of the gut microbiota indicated that after the administration of heat-killed A. oryzae spores, the relative abundance of an anti-inflammatory Bifidobacterium pseudolongum strain became 2.0-fold greater than that of the control. Next, we examined the effect of A. oryzae spore administration on the development of colitis induced by dextran sodium sulfate in mice; we found that colitis was alleviated by not only heat-killed A. oryzae spores, but also the cell wall extracted from the spores. Our findings suggest that A. oryzae holds considerable potential for commercial application in the production of both traditional Japanese fermented foods and new foods with prebiotic functions.

Frequency and Clinicopathological Characteristics of Patients With KRAS/BRAF Double-Mutant Colorectal Cancer: An In Silico Study.

KRAS and BRAF mutations are currently thought to be mutually exclusive as their co-occurrence is extremely rare. Therefore, clinicopathological and molecular characteristics of colorectal carcinoma with KRAS/BRAF double mutations are unclear. We aimed to investigate the frequency and clinicopathological characteristics of double-mutant colorectal carcinoma and its differences from KRAS/BRAF single-mutant colorectal carcinoma using bioinformatics tools. We estimated the KRAS/BRAF double mutation frequency in the whole exon and coding sequences via bioinformatic analyses of three datasets from cBioPortal. We compared the clinicopathological characteristics, microsatellite instability status, BRAF classification, and tumor mutation burden of patients harboring the double mutants with those of patients harboring KRAS or BRAF single mutations. We integrated three large datasets and found that the frequency of the KRAS/BRAF double mutation in the dataset was 1.2% (29/2347). The double mutation occurred more frequently in males, with a slightly higher occurrence in the right side of the colon. Sex, histological type, histological grade, microsatellite instability, and tumor mutation burden of the patients harboring KRAS-mutant, BRAF-mutant, and double-mutant colorectal carcinoma varied significantly. The frequency of double-mutant colorectal carcinoma was 60 times higher than that previously reported. Significantly fewer double-mutant colorectal carcinoma cases were classified as BRAF class 1 and more were classified as unknown. Our findings indicate that the biological characteristics of double-mutant tumors are different from those of single-mutant tumors.

WUSCHEL-related homeobox family genes in rice control lateral root primordium size.

The development of a plastic root system is essential for stable crop production under variable environments. Rice plants have two types of lateral roots (LRs): S-type (short and thin) and L-type (long, thick, and capable of further branching). LR types are determined at the primordium stage, with a larger primordium size in L-types than S-types. Despite the importance of LR types for rice adaptability to variable water conditions, molecular mechanisms underlying the primordium size control of LRs are unknown. Here, we show that two WUSCHEL-related homeobox (WOX) genes have opposing roles in controlling LR primordium (LRP) size in rice. Root tip excision on seminal roots induced L-type LR formation with wider primordia formed from an early developmental stage. QHB/OsWOX5 was isolated as a causative gene of a mutant that is defective in S-type LR formation but produces more L-type LRs than wild-type (WT) plants following root tip excision. A transcriptome analysis revealed that OsWOX10 is highly up-regulated in L-type LRPs. OsWOX10 overexpression in LRPs increased the LR diameter in an expression-dependent manner. Conversely, the mutation in OsWOX10 decreased the L-type LR diameter under mild drought conditions. The qhb mutants had higher OsWOX10 expression than WT after root tip excision. A yeast one-hybrid assay revealed that the transcriptional repressive activity of QHB was lost in qhb mutants. An electrophoresis mobility shift assay revealed that OsWOX10 is a potential target of QHB. These data suggest that QHB represses LR diameter increase, repressing OsWOX10 Our findings could help improve root system plasticity under variable environments.

Development of a Biocontained Toluene-Degrading Bacterium for Environmental Protection.

Biocontainment is a safeguard strategy for preventing uncontrolled proliferation of genetically engineered microorganisms (GEMs) in the environment. Biocontained GEMs are designed to survive only in the presence of a specific molecule. The design of a pollutant-degrading and pollutant-dependent GEM prevents its proliferation after cleaning the environment. In this study, we present a biocontained toluene-degrading bacterium based on Acinetobacter sp. Tol 5. The bamA gene, which encodes an essential outer membrane protein, was deleted from the chromosome of Tol 5 but complemented with a plasmid carrying a bamA gene regulated by the Pu promoter and the regulatory protein XylR. The resultant strain (PuBamA) degraded toluene, similarly to the wild-type Tol 5. Although the cell growth of the PuBamA strain was remarkably inhibited after toluene depletion, escape mutants emerged at a frequency of 1 per 5.3 × 10-7 cells. Analyses of escape mutants revealed that insertion sequences (ISs) carrying promoters were inserted between the Pu promoter and the bamA gene on the complemented plasmid. MinION deep sequencing of the plasmids extracted from the escape mutants enabled the identification of three types of ISs involved in the emergence of escape mutants, suggesting a strategy for reducing it. IMPORTANCE GEMs are beneficial for various applications, including environmental protection. However, the risks of GEM release into the environment have been debated for a long time. If a pollutant is employed as a specific molecule for a biocontainment system, GEMs capable of degrading pollutants are available for environmental protection. Nevertheless, to our knowledge, biocontained degraders for real pollutants have not been reported in academic journals so far. This is possibly due to the difficulty in the expression of enzymes for degrading pollutants in a tractable bacterium such as Escherichia coli. On the other hand, bacteria with enzymes for degrading pollutants are often intractable as a host of GEMs due to the shortage of tools for genetic manipulation. This study reports the feasibility of a biocontainment strategy for a toluene degrader. Our results provide useful insights into the construction of a GEM biocontainment system for environmental protection.

Clinicopathological Features, Tumor Mutational Burden, and Tumour-Infiltrating Lymphocyte Interplay in ERBB2-Mutated Breast Cancer: In Silico Analysis.

Recent evidence suggests that somatic mutations in ERBB2 activate ERBB2 signaling. These mutations occur at a frequency of approximately 3% in breast cancer (BC). ERBB2 mutations indicate poor prognosis as they are associated with recurrence and metastasis. This study aimed to evaluate the clinicopathological features, immune infiltration levels, tumor mutational burden (TMB), and tumor-infiltrating lymphocytes (TILs) in ERBB2-mutated breast cancer (ERBB2-mutated BC) using a bioinformatic approach and publicly available datasets (i.e., TCGA-BRCA and TIMER2.0). ERBB2-mutated BCs were associated with a high histological grade. ERBB2-mutated BCs comprised invasive breast carcinoma of no special type (21/35, 60%), classic invasive lobular carcinoma (12/35, 34.3%), and pleomorphic invasive lobular carcinoma (2/35, 5.7%). A Kaplan-Meier survival curve demonstrated that ERBB2-mutated BC was associated with a significantly worse prognosis compared to ERBB2 non-mutated BC (p < 0.01). Furthermore, 40% (14/35) of the patients with ERBB2-mutated BC harbored CDH1 mutations. Mutations at L755 and V777 accounted for 30.5% of these mutations in ERBB2-mutated BC, suggesting that these sites are mutational hot spots in BC, particularly in invasive lobular carcinoma. Of the ERBB2-mutated BCs, 8.6% were classified as TIL-high, whereas 77.1% were TILs-low; TMB significantly correlated with TILs (p < 0.05). CD8+ T cell infiltration levels were significantly higher in ERBB2 non-mutated BC. Among ERBB2-mutated BCs, 22.9% were classified as TMB-high, which was significantly higher than the rate in the ERBB2 non-mutated BC (p < 0.01). These findings provide evidence for a link between ERBB2 mutations and high TMB in BC.

Mutation of OUR1/OsbZIP1, which encodes a member of the basic leucine zipper transcription factor family, promotes root development in rice through repressing auxin signaling.

A well-developed root system is essential for efficient water uptake, particularly in drought-prone environments. However, the molecular mechanisms underlying the promotion of root development are poorly understood. We identified and characterized a rice mutant, outstanding rooting1 (our1), which exhibited a well-developed root system. The our1 mutant displayed typical auxin-related phenotypes, including elongated seminal root and defective gravitropism. Seminal root elongation in the our1 mutant was accelerated via the promotion of cell division and elongation. In addition, compared with the wild type, the density of short and thin lateral roots (S-type LRs) was reduced in the our1 mutant, whereas that of long and thick LRs (L-type LRs) was increased. Expression of OUR1, which encodes OsbZIP1, a member of the basic leucine zipper transcription factor family, was observed in the seminal root tip and sites of LR emergence, wherein attenuation of reporter gene expression levels controlled by the auxin response promoter DR5 was also observed in the our1 mutant. Taken together, our results indicate that the our1 gene promotes root development by suppressing auxin signaling, which may be a key factor contributing to an improvement in root architecture.

High sucrose diet-induced dysbiosis of gut microbiota promotes fatty liver and hyperlipidemia in rats.

Excess sucrose intake has been found to be a major factor in the development of metabolic syndrome, especially in promoting nonalcoholic fatty liver disease. The excess fructose is believed to targets the liver to promote de novo lipogenesis, as described in major biochemistry textbooks. On the contrary, in this study, we explored the possible involvement of gut microbiota in excess sucrose-induced lipid metabolic disorders, to validate a novel mechanism by which excess sucrose causes hepatic lipid metabolic disorders via alterations to the gut microbial community structure. Wistar male rats were fed either a control starch diet or a high-sucrose diet for 4 weeks. Half of the rats in each group were treated with an antibiotic cocktail delivered via drinking water for the entire experimental period. After 4 weeks, rats fed with the high-sucrose diet showed symptoms of fatty liver and hyperlipidemia. The architecture of cecal microbiota was altered in rats fed with high-sucrose diet as compared to the control group, with traits including increased ratios of the phyla Bacteroidetes/Firmicutes, reduced α-diversity, and diurnal oscillations changes. Antibiotic administration rescued high-sucrose diet-induced lipid accumulation in the both blood and liver. Levels of two microbial metabolites, formate and butyrate, were reduced in rats fed with the high-sucrose diet. These volatile short-chain fatty acids might be responsible for the sucrose-induced fatty liver and hyperlipidemia. Our results indicate that changes in the gut microbiota induced by a high-sucrose diet would promote the development of nonalcoholic fatty liver disease via its metabolites, such as short-chain fatty acids.

Increasing cell-free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA-binding proteins.

In crude extract-based cell-free protein synthesis (CFPS), DNA templates are transcribed and translated into functional proteins. Although linear expression templates (LETs) are less laborious and expensive to generate, plasmid templates are often desired over polymerase chain reaction-generated LETs due to increased stability and protection against exonucleases present in the extract of the reaction. Here we demonstrate that addition of a double stranded DNA-binding protein to the CFPS reaction, termed single-chain Cro protein (scCro), achieves terminal protection of LETs. This CroP-LET (scCro-based protection of LET) method effectively increases superfolder green fluorescent protein (sfGFP) expression levels from LETs in Escherichia coli CFPS reactions by sixfold. Our yields are comparable to other strategies that provide chemical and enzymatic DNA stabilization in E. coli CFPS. Notably, we also report that the CroP-LET method successfully enhanced yields in CFPS platforms derived from nonmodel organisms. Our results show that CroP-LET increased sfGFP yields by 18-fold in the Vibrio natriegens CFPS platform. With the fast-expanding applications of CFPS platforms, this method provides a practical and generalizable solution to protect linear expression DNA templates.

Ultra-high-throughput analysis of functional biomolecules using in vitro selection and bioinformatics.

Functional analysis of biomolecules, including nucleic acids and proteins, is important for understanding biological mechanisms in living cells such as gene expression and metabolism. To analyze diverse biomolecular functions, large-scale screening systems for biomolecules have been developed for various applications such as to improve enzyme activity and identify target binding molecules. One of these systems, the Bead Display system, utilizes emulsion technology and is a powerful tool for rapidly screening functional nucleic acids or proteins in vitro. Furthermore, an analytical pipeline that consists of genomic systematic evolution of ligands by exponential enrichment (gSELEX)-Seq, gene expression analysis, and bioinformatics was shown to be a robust platform for comprehensively identifying genes regulated by a transcription factor. This review provides an overview of the biomolecular screening methods developed to date.

Development of a dual monoclonal antibody sandwich enzyme-linked immunosorbent assay for the detection of swine influenza virus using rabbit monoclonal antibody by Ecobody technology.

A dual monoclonal antibody sandwich enzyme-linked immunosorbent assay (mAb sandwich ELISA) has been developed using rabbit monoclonal antibodies generated by Ecobody technology, which includes the isolation of single B cells binding to a specific antigen, amplification of the heavy and light chains of these immunoglobulins, and expression of the fragment of antigen binding (Fab) by cell-free protein synthesis (CFPS). A rabbit was immunized with swine influenza virus (SIV) vaccine, from which single B cells binding to the antigen were isolated. Then, immunoglobulin mRNA was amplified from single cells by reverse transcription-polymerase chain reaction, followed by the attachment of a T7 promoter, appropriate tags, and a T7 terminator for the expression of the Fab portion by CFPS. By taking advantage of two different peptide tags fused to the same Fab, optimal combinations for coating Fab on assay plates and detecting Fab, both synthesized by CFPS, were investigated for mAb sandwich ELISA. Pairs of Fab detected 0.5 ng SIV in the assay. In summary, this result showed the applicability of Ecobody technology for a variety of immunodetection kits for high throughput analyses.