Antimicrobial agents have eradicated many infectious diseases and significantly improved our living environment. However, abuse of antimicrobial agents has accelerated the emergence of ...multidrug-resistant microorganisms, and there is an urgent need for novel antibiotics. Antimicrobial peptides (AMPs) have attracted attention as a novel class of antimicrobial agents because AMPs efficiently kill a wide range of species, including bacteria, fungi, and viruses, via a novel mechanism of action. In addition, they are effective against pathogens that are resistant to almost all conventional antibiotics. AMPs have promising properties; they directly disrupt the functions of cellular membranes and nucleic acids, and the rate of appearance of AMP-resistant strains is very low. However, as pharmaceuticals, AMPs exhibit unfavorable properties, such as instability, hemolytic activity, high cost of production, salt sensitivity, and a broad spectrum of activity. Therefore, it is vital to improve these properties to develop novel AMP treatments. Here, we have reviewed the basic biochemical properties of AMPs and the recent strategies used to modulate these properties of AMPs to enhance their safety.
Despite the importance of type I interferon (IFN-I) in systemic lupus erythematosus (SLE) pathogenesis, the mechanisms of IFN-I production have not been fully elucidated. Recognition of nucleic acids ...by DNA sensors induces IFN-I and interferon-stimulated genes (ISGs), but the involvement of cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) and stimulator of interferon genes (STING) in SLE remains unclear. We studied the role of the cGAS-STING pathway in the IFN-I-producing cascade driven by SLE serum.
We collected sera from patients with SLE (n=64), patients with other autoimmune diseases (n=31) and healthy controls (n=35), and assayed them using a cell-based reporter system that enables highly sensitive detection of IFN-I and ISG-inducing activity. We used Toll-like receptor-specific reporter cells and reporter cells harbouring knockouts of cGAS, STING and IFNAR2 to evaluate signalling pathway-dependent ISG induction.
IFN-I bioactivity and ISG-inducing activities of serum were higher in patients with SLE than in patients with other autoimmune diseases or healthy controls. ISG-inducing activity of SLE sera was significantly reduced in STING-knockout reporter cells, and STING-dependent ISG-inducing activity correlated with disease activity. Double-stranded DNA levels were elevated in SLE. Apoptosis-derived membrane vesicles (AdMVs) from SLE sera had high ISG-inducing activity, which was diminished in cGAS-knockout or STING-knockout reporter cells.
AdMVs in SLE serum induce IFN-I production through activation of the cGAS-STING pathway. Thus, blockade of the cGAS-STING axis represents a promising therapeutic target for SLE. Moreover, our cell-based reporter system may be useful for stratifying patients with SLE with high ISG-inducing activity.
Background
Neural networks generate complex behaviors. However, the relationships between them have not been understood well. Thus, we have developed ‘functional cellomics’, which enables functional ...annotation of neural networks in a hypothesis‐free manner. The key is the stochastic labeling of opsin achieved by Cre‐mediated stochastic recombination. We designed a gene cassette, which contained two sets of lox variants, lox2272 and loxP, alternately. A transcription factor, QF2w, is interposed between loxP. Cre excises DNA with an exclusive choice of between lox2272 or loxP. When Cre excises lox2272, expressed QF2w induces opsin. Constructing a C. elegans strain carrying this gene cassette, we successfully implemented the stochastic labeling of opsin dependent on Cre induction 1. However, the labeling rate of opsin was about 30 %. If a large subset of neurons is labeled by opsin, it becomes difficult to distinguish the function of each neuron. In this study, we demonstrated a strategy to precisely control the labeling rate of opsin by machine learning‐guided engineering of Cre‐lox recombination.
Method
In our genetic circuit, the opsin coding gene is expressed in neurons where Cre‐lox recombination occurs between lox2272 sequences. Therefore, we hypothesized that the labeling rate of opsin would be reduced by using lox2272 variants, which is difficult to be excised by Cre. Thus, we generated a library of randomized lox sequences by PCR and introduced them in Saccharomyces cerevisiae. We then induced Cre in the S. cerevisiae and extracted DNA from S. cerevisiae. We used NGS to assess the excision rate of the lox2272 variants. To develop a machine learning model to predict the excision rate of the lox2272 variants, we trained a Gaussian process (GP) model using data containing lox2272 sequences and their excision rates by Cre. Approximately 1000 lox2272 variants were randomly selected as training data and 100 variants were selected to test the prediction accuracy.
Results and Discussion
We quantified the excision rates of over 2000 lox2272 variants by NGS and successfully found lox2272 variants with excision efficiencies ranging from 0.05% to 100%. The results of the NGS analysis were confirmed by qPCR. Next, we created a GP model and observed a high correlation between the actual and predicted cleavage rates of the test data. Using the GP model, we predicted the efficiency of the unevaluated lox2272 sequence and successfully identified lox2272 variants that had various cleavage rates. These results demonstrate the feasibility of precise control of opsin labeling rates in functional cellomics 2.
1 Aoki et al, Sci Rep, 8, 10380, 2018
2 Yamauchi et al, submitted
Artificial cells containing in vitro transcription and translation (IVTT) systems inside liposomes are important for the reconstruction and analysis of various biological systems. To improve the ...accessibility of artificial cell research, it is important that artificial cells can be constructed using only commercially available components. Here, we optimized the construction of artificial cells containing PUREfrex2.0, a commercially available IVTT with high transcriptional and translational activity. Specifically, the composition of the inner and outer s olutions of the liposomes and the concentrations of lipids, glucose/sucrose, potassium glutamate, and magnesium acetate were systematically optimized, and finally we found a protocol for the stable construction of artificial cells containing PUREfre×2.0. These findings are expected to be important in expanding the artificial cell research community.
Abstract
Yeast cell surface display (YSD) has been used to engineer various proteins, including antibodies. Directed evolution, which subjects a gene to iterative rounds of mutagenesis, selection and ...amplification, is useful for protein engineering. In vivo continuous mutagenesis, which continuously diversifies target genes in the host cell, is a promising tool for accelerating directed evolution. However, combining in vivo continuous evolution and YSD is difficult because mutations in the gene encoding the anchor proteins may inhibit the display of target proteins on the cell surface. In this study, we have developed a modified YSD method that utilises SpyTag/SpyCatcher-based in vivo protein ligation. A nanobody fused with a SpyTag of 16 amino acids and an anchor protein fused with a SpyCatcher of 113 amino acids are encoded by separate gene cassettes and then assembled via isopeptide bond formation. This system achieved a high display efficiency of more than 90%, no intercellular protein ligation events, and the enrichment of target cells by cell sorting. These results suggested that our system demonstrates comparable performance with conventional YSD methods; therefore, it can be an appropriate platform to be integrated with in vivo continuous evolution.
The creation of a self-replicating synthetic cell is an essential to understand life self-replication. One method to create self-replicating artificial cells is to reconstitute the self-replication ...system of living organisms in vitro. In a living cell, self-replication is achieved via a system called the autonomous central dogma, a system in which central dogma-related factors are autonomously synthesized and genome replication, transcription, and translation are driven by nascent factors. Various studies to reconstitute some processes of the autonomous central dogma in vitro have been conducted. However, in vitro reconstitution of the entire autonomous central dogma system is difficult as it requires balanced expression of several related genes. Therefore, we developed a method to simultaneously quantify and optimize the in vitro expression balance of multiple genes. First, we developed a quantitative mass spectrometry method targeting genome replication-related proteins as a model of central dogma-related factors and acquired in vitro expression profiles of these genes. Additionally, we demonstrated that the in vitro expression balance of these genes can be easily optimized by adjusting the input gene ratio based on the data obtained by the developed method. This study facilitated the easy optimization of the in vitro expression balance of multiple genes. Therefore, extending the scope of this method to other central dogma-related factors will accelerate attempts of self-replicating synthetic cells creation.
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Cancer treatment has been revolutionized by immune checkpoint inhibitors, which regulate immune cell function by blocking the interactions between immune checkpoint molecules and their ligands. The ...interaction between programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) is a target for immune checkpoint inhibitors. Nanobodies, which are recombinant variable domains of heavy-chain-only antibodies, can replace existing immune checkpoint inhibitors, such as anti-PD-1 or anti-PD-L1 conventional antibodies. However, the screening process for high-affinity nanobodies is laborious and time-consuming. Here, we identified high-affinity anti-PD-1 nanobodies using peptide barcoding, which enabled reliable and efficient screening by distinguishing each nanobody with a peptide barcode that was genetically appended to each nanobody. We prepared a peptide-barcoded nanobody (PBNb) library with thousands of variants. Three high-affinity PBNbs were identified from the PBNb library by quantifying the peptide barcodes derived from high-affinity PBNbs. Furthermore, these three PBNbs neutralized the interaction between PD-1 and PD-L1. Our results demonstrate the utility of peptide barcoding and the resulting nanobodies can be used as experimental tools and antitumor agents.
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Antibiotics have significantly improved our living environments. However, overuse of antibiotics has led to the emergence of multi-drug resistant microorganisms, and the subsequent constant demand ...for the exploration of novel antibiotics. To this end, antimicrobial peptides (AMPs) have attracted much attention as a novel class of antibiotics. AMPs have strong antimicrobial activity against a wide-range of species, including gram-positive and gram-negative bacteria, fungi, and viruses. In addition, they are also effective against pathogenic organisms that are resistant to conventional drugs. Despite their great potential, the hemolytic activity and a highly broad spectrum of activity of AMPs dictate the need for amendments to develop safe pharmaceuticals. The human body contains commensal microflora as an integral part of complex mucosal surfaces that offers protection against pathogenic organisms. Administration of antibiotics with broad spectra of activity disrupts the indigenous microflora and increases the risks of diarrhea and other fatal infections. Therefore, it is difficult, but vital, to develop treatments capable of rapidly eliminating pathogenic organisms while maintaining the commensal microbiota. As such, novel pharmaceuticals, safe designer AMPs have been heavily researched. In this article, we review recent attempts to spatially and temporally regulate AMPs to enhance the quality-of-life of patients.
Cruciferous vegetables are rich sources of glucosinolates (GSLs). GSLs are degraded into isothiocyanates, which are potent anticarcinogens, by human gut bacteria. However, the mechanisms and enzymes ...involved in gut bacteria-mediated GSL metabolism are currently unclear. This study aimed to elucidate the enzymes involved in GSL metabolism in lactic acid bacteria, a type of gut bacteria. Companilactobacillus farciminis KB1089 was selected as a lactic acid bacteria strain model that metabolizes sinigrin, which is a GSL, into allylisothiocyanate. The sinigrin-metabolizing activity of this strain is induced under glucose-absent and sinigrin-present conditions. A quantitative comparative proteomic analysis was conducted and a total of 20 proteins that were specifically expressed in the induced cells were identified. Three candidate proteins, β-glucoside-specific IIB, IIC, IIA phosphotransferase system (PTS) components (CfPttS), 6-phospho-β-glucosidase (CfPbgS) and a hypothetical protein (CfNukS), were suspected to be involved in sinigrin-metabolism and were thus investigated further. We hypothesize a pathway for sinigrin degradation, wherein sinigrin is taken up and phosphorylated by CfPttS, and subsequently, the phosphorylated entity is degraded by CfPbgS. As expression of both pttS and pbgS genes clearly gave Escherichia coli host strain sinigrin converting activity, these genes were suggested to be responsible for sinigrin degradation. Furthermore, heterologous expression analysis using Lactococcus lactis suggested that CfPttS was important for sinigrin degradation and CfPbgS degraded phosphorylated sinigrin.
The coral microbiome has attracted increased attention because of its potential roles in host protection against deadly diseases. However, little is known about the role of coral-associated bacteria ...against the temperature-dependent opportunistic pathogen
Vibrio coralliilyticus
. In this study, we tested whether bacteria associated with the reef-building coral
Galaxea fascicularis
could inhibit the growth of
V. coralliilyticus
. Twenty-nine cultivable bacteria were successfully isolated from a healthy colony of
G. fascicularis
kept in an aquarium. Among the bacterial isolates, three
Ruegeria
sp. strains inhibited the growth of
V. coralliilyticus
P1 as a reference strain and
Vibrio
sp. isolated in this study.
Ruegeria
sp. strains were also detected from other
G. fascicularis
colonies in the aquarium and in previous field studies by 16S rRNA amplicon sequencing, suggesting that
Ruegeria
sp. strains are common among
G. fascicularis
colonies. These results illuminate the potential role of
Ruegeria
sp. in protecting corals against pathogenic
Vibrio
species.
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