Cell-Free Synthesis of Human Endothelin Receptors and Its Application to Ribosome Display.

Engineering G-protein-coupled receptors (GPCRs) for improved stability or altered function is of great interest, as GPCRs consist of the largest protein family, are involved in many important signaling pathways, and thus, are one of the major drug targets. Here, we report the development of a high-throughput screening method for GPCRs using a reconstituted in vitro transcription-translation (IVTT) system. Human endothelin receptor type-B (ETBR), a class A GPCR that binds endothelin-1 (ET-1), a 21-residue peptide hormone, was synthesized in the presence of nanodisc (ND) composed of a phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG). The ET-1 binding of ETBR was significantly reduced or was undetectable when other phospholipids were used for ND preparation. However, when functional ETBR purified from Sf9 cells was reconstituted into NDs, ET-1 binding was observed with two different phospholipids tested, including POPG. These results suggest that POPG likely supports the folding of ETBR into its functional form in the IVTT system. Using the same conditions as ETBR, whose three-dimensional structure has been solved, human endothelin receptor type-A (ETAR), whose three-dimensional structure remains unsolved, was also synthesized in its functional form. By adding POPG-ND to the IVTT system, both ETAR and ETBR were successfully subjected to ribosome display, a method of in vitro directed evolution that facilitates the screening of up to 1012 mutants. Finally, using a mock library, we showed that ribosome display can be applied for gene screening of ETBR, suggesting that high-throughput screening and directed evolution of GPCRs is possible in vitro.

Antisense Oligonucleotide Modified with Disulfide Units Induces Efficient Exon Skipping in mdx Myotubes through Enhanced Membrane Permeability and Nucleus Internalization.

We have found that antisense oligonucleotides and siRNA molecules modified with repeat structures of disulfide units can be directly introduced into the cytoplasm and exhibit a suppressive effect on gene expression. In this study, we analyzed the mechanism of cellular uptake of these membrane-permeable oligonucleotides (MPONs). Time-course analysis by confocal microscopy showed that the uptake of MPONs from the plasma membrane to the cytoplasm reached 50 % of the total uptake in about 5 min. In addition, analysis of the plasma membrane proteins to which MPONs bind, identified several proteins, including voltage-dependent anion channel. Next, we analyzed the behavior of MPONs in the cell and found them to be abundant in the nucleus as early as 24 h after addition with the amount increasing further after 48 and 72 h. The amount of MPONs was 2.5-fold higher than that of unmodified oligonucleotides in the nucleus after 72 h. We also designed antisense oligonucleotides and evaluated the effect of MPONs on mRNA exon skipping using DMD model cells; MPONs caused exon skipping with 69 % efficiency after 72 h, which was three times higher than the rate of the control. In summary, the high capacity for intracytoplasmic and nuclear translocation of MPONs is expected to be useful for therapeutic strategies targeting exon skipping.

Effects of chronic intracerebroventricular infusion of neurosecretory protein GL on body mass and food and water intake in chicks.

Recently, we discovered a novel cDNA encoding the precursor of a small secretory protein, neurosecretory protein GL (NPGL), in the chicken mediobasal hypothalamus. In this study, immunohistochemical analysis revealed that NPGL was produced in the infundibular and medial mammillary nuclei of the mediobasal hypothalamus, with immunoreactive fibers also detected in the hypothalamus and the median eminence. As it is known that these regions are involved in feeding behavior in chicks, we surveyed the effects of chronic intracerebroventricular infusion of NPGL on feeding behavior and body mass for a period of two weeks. NPGL stimulated food and water intake, with a concomitant increase in body mass. However, NPGL did not influence mRNA expression of several hypothalamic ingestion-related neuropeptides. Our data suggest that NPGL may be a novel neuronal regulator involved in growth processes in chicks.

Microwave-assisted solid-phase peptide synthesis of neurosecretory protein GL composed of 80 amino acid residues.

We recently identified a novel cDNA encoding a small secretory protein of 80 amino acid residues, termed neurosecretory protein GL (NPGL), from the chicken hypothalamus. Homologs of NPGL have been reported to be present in mammals, such as human and rat. NPGL is amidated at its C-terminus, contains an intramolecular disulfide bond, and is hydrophobic in nature. In this study, we have optimized the synthesis of the entire 80-amino acid peptide sequence of rat NPGL by microwave-assisted solid-phase peptide synthesis. NPGL was obtained with a 10% yield when the coupling reactions were performed using 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxid hexafluorophosphate (HATU) at 50 °C for 5 min, and Fmoc deprotections were performed using 40% piperidine containing 0.1 M HOBt. Furthermore, the disulfide bond of NPGL was formed with 20% yield with the use of glutathione-containing redox buffer and 50% acetonitrile.

Identification of a cDNA encoding a novel small secretory protein, neurosecretory protein GL, in the chicken hypothalamic infundibulum.

To find novel neuropeptide and/or peptide hormone precursors in the avian brain, we performed a cDNA subtractive screen of the chicken hypothalamic infundibulum, which contains one of the feeding and neuroendocrine centers. After sequencing 596 clones, we identified a novel cDNA encoding a previously unknown protein. The deduced precursor protein consisted of 182 amino acid residues, including one putative small secretory protein of 80 amino acid residues. This small protein was flanked at the N-terminus by a signal peptide and at the C-terminus by a glycine amidation signal and a dibasic amino acid cleavage site. Because the predicted C-terminal amino acids of the small protein were Gly-Leu-NH2, the small protein was named neurosecretory protein GL (NPGL). Quantitative RT-PCR analysis demonstrated specific expression of the NPGL precursor mRNA in the hypothalamic infundibulum. Furthermore, the mRNA levels in the hypothalamic infundibulum increased during post-hatching development. In situ hybridization analysis showed that the cells containing the NPGL precursor mRNA were localized in the medial mammillary nucleus and infundibular nucleus within the hypothalamic infundibulum of 8- and 15-day-old chicks. Subcutaneous infusion of NPGL in chicks increased body weight gain without affecting food intake. To our knowledge, this is the first report to describe the identification and localization of the NPGL precursor mRNA and the function of its translated product in animals. Our findings indicate that NPGL may participate in the growth process in chicks.

Identification of neurotensin and LANT-6 and localization of mRNA encoding their precursor in the chicken brain.

Neurotensin (NT) and neurotensin-related peptide (Lys(8), Asn(9), NT(8-13): LANT-6) have previously been purified from chicken intestine. However, the presence of these peptides and the localization of their precursor mRNA in the brain were not well understood. In the present study, through a comprehensive analysis of bioactive substances, NT and LANT-6 were identified in the chicken brain using tandem mass spectrometry combined with a bioassay of the colon contraction. The effect of NT and LANT-6 on the colon contraction was assessed, and NT was found to be 10 times more potent than LANT-6. Furthermore, the sites of NT/LANT-6 precursor mRNA expression in the brain were investigated using quantitative RT-PCR. The result showed that the mRNA was expressed most in the telencephalon, followed by the diencephalon. In situ hybridization analysis revealed that cells containing NT/LANT-6 precursor mRNA were widely distributed throughout the brain except for the cerebellum. Additionally, these were highly concentrated in the frontal telencephalon, including the nidopallium, hyperpallium, and hippocampus. Collectively, these results indicate that NT and LANT-6 are produced in the chicken brain, and they may participate in multiple functions.

Fibrinopeptide A-induced blood-feeding arrest in the yellow fever mosquito Aedes aegypti.

Female mosquitoes engage in blood feeding from their hosts to facilitate egg maturation but cease feeding once a sufficient blood meal has been acquired. Abdominal distention has been proposed as a contributing factor; however, it has also been suggested that there are chemical controls. In this study, we focus on negative chemical regulators of blood feeding, particularly those present in the host blood. Serum derived from animal blood inhibits the feeding of ATP, a phagostimulant of blood feeding in Aedes aegypti. Fibrinopeptide A (FPA), a 16-amino acid peptide cleaved from fibrinogen during blood coagulation, serves as an inhibitory factor in the serum. Our findings suggest that blood-feeding arrest in female mosquitoes is triggered by the detection of FPA in the host blood, which increases as blood coagulation proceeds in the mosquito's midgut, highlighting the role of host-derived substances as negative regulators of mosquito behavior.

The usefulness of machine-learning-based evaluation of clinical and pretreatment 18F-FDG-PET/CT radiomic features for predicting prognosis in patients with laryngeal cancer.

OBJECTIVE: To examine whether machine learning (ML) analyses involving clinical and 18F-FDG-PET-based radiomic features are helpful in predicting prognosis in patients with laryngeal cancer. METHODS: This retrospective study included 49 patients with laryngeal cancer who underwent18F-FDG-PET/CT before treatment, and these patients were divided into the training (n = 34) and testing (n = 15) cohorts.Seven clinical (age, sex, tumor size, T stage, N stage, Union for International Cancer Control stage, and treatment) and 40 18F-FDG-PET-based radiomic features were used to predict disease progression and survival. Six ML algorithms (random forest, neural network, k-nearest neighbors, naïve Bayes, logistic regression, and support vector machine) were used for predicting disease progression. Two ML algorithms (cox proportional hazard and random survival forest [RSF] model) considering for time-to-event outcomes were used to assess progression-free survival (PFS), and prediction performance was assessed by the concordance index (C-index). RESULTS: Tumor size, T stage, N stage, GLZLM_ZLNU, and GLCM_Entropy were the five most important features for predicting disease progression.In both cohorts, the naïve Bayes model constructed by these five features was the best performing classifier (training: AUC = 0.805; testing: AUC = 0.842). The RSF model using the five features (tumor size, GLZLM_ZLNU, GLCM_Entropy, GLRLM_LRHGE and GLRLM_SRHGE) exhibited the highest performance in predicting PFS (training: C-index = 0.840; testing: C-index = 0.808). CONCLUSION: ML analyses involving clinical and 18F-FDG-PET-based radiomic features may help predict disease progression and survival in patients with laryngeal cancer. ADVANCES IN KNOWLEDGE: ML approach using clinical and 18F-FDG-PET-based radiomic features has the potential to predict prognosis of laryngeal cancer.

A large-scale targeted proteomics of plasma extracellular vesicles shows utility for prognosis prediction subtyping in colorectal cancer.

PURPOSE: The pathogenesis of cancers depends on the molecular background of each individual patient. Therefore, verifying as many biomarkers as possible and clarifying their relationships with each disease status would be very valuable. We performed a large-scale targeted proteomics analysis of plasma extracellular vesicles (EVs) that may affect tumor progression and/or therapeutic resistance. EXPERIMENTAL DESIGN: Plasma EVs from 59 were collected patients with colorectal cancer (CRC) and 59 healthy controls (HC) in cohort 1, and 150 patients with CRC in cohort 2 for the large-scale targeted proteomics analysis of 457 proteins as candidate CRC markers. The Mann-Whitney-Wilcoxon test and random forest model were applied in cohort 1 to select promising markers. Consensus clustering was applied to classify patients with CRC in cohort 2. The Kaplan-Meier method and Cox regression analysis were performed to identify potential molecular factors contributing to the overall survival (OS) of patients. RESULTS: In the analysis of cohort 1, 99 proteins were associated with CRC. The analysis of cohort 2 revealed two clusters showing significant differences in OS (p = 0.017). Twelve proteins, including alpha-1-acid glycoprotein 1 (ORM1), were suggested to be associated with the identified CRC subtypes, and ORM1 was shown to significantly contribute to OS, suggesting that ORM1 might be one of the factors closely related to the OS. CONCLUSIONS: The study identified two novel subtypes of CRC, which exhibit differences in OS, as well as important biomarker proteins that are closely related to the identified subtypes. Liquid biopsy assessment with targeted proteomics analysis was proposed to be crucial for predicting the CRC prognosis.

Capillary microsampling-based single-cell metabolomics by mass spectrometry and its applications in medicine and drug discovery.

Characterization of cellular metabolic states is a technical challenge in biomedicine. Cellular heterogeneity caused by inherent diversity in expression of metabolic enzymes or due to sensitivity of metabolic reactions to perturbations, necessitates single cell analysis of metabolism. Heterogeneity is typically seen in cancer and thus, single-cell metabolomics is expectedly useful in studying cancer progression, metastasis, and variations in cancer drug response. However, low sample volumes and analyte concentrations limit detection of critically important metabolites. Capillary microsampling-based mass spectrometry approaches are emerging as a promising solution for achieving single-cell omics. Herein, we focus on the recent advances in capillary microsampling-based mass spectrometry techniques for single-cell metabolomics. We discuss recent technical developments and applications to cancer medicine and drug discovery.

Versatile and multiplexed mass spectrometry-based absolute quantification with cell-free-synthesized internal standard peptides.

Preparation of stable isotope-labeled internal standard peptides is crucial for mass spectrometry (MS)-based targeted proteomics. Herein, we developed versatile and multiplexed absolute protein quantification method using MS. A previously developed method based on the cell-free peptide synthesis system, termed MS-based quantification by isotope-labeled cell-free products (MS-QBiC), was improved for multiple peptide synthesis in one-pot reaction. We pluralized the quantification tags used for the quantification of synthesized peptides and thus, made it possible to use cell-free synthesized isotope-labeled peptides as mixtures for the absolute quantification. The improved multiplexed MS-QBiC method was proved to be applied to clarify ribosomal proteins stoichiometry in the ribosomal subunit, one of the largest cellular complexes. The study demonstrates that the developed method enables the preparation of several dozens and even several hundreds of internal standard peptides within a few days for quantification of multiple proteins with only a single-run of MS analysis. SIGNIFICANCE: The developed method can be applied for the preparation of internal standard peptides without limiting the number of peptides to be synthesized, which may result in more practical screening of quantitatively reliable peptides, one of the fundamental steps in the reliable absolute quantification using MS. Furthermore, the method is highly versatile for proteome analysis of any organisms or species without any cDNA or SIL peptide libraries. The quantification can be finished in a few days including design and preparation of appropriate SIL peptides using small-scale batch cell-free reactions, which has a potential to be a part of the standard methodology in a field of quantitative proteomics.

In vitro reconstitution of the Escherichia coli 70S ribosome with a full set of recombinant ribosomal proteins.

Many studies of the reconstitution of the Escherichia coli small ribosomal subunit from its individual molecular parts have been reported, but contrastingly, similar studies of the large ribosomal subunit have not been well performed to date. Here, we describe protocols for preparing the 33 ribosomal proteins of the E. coli 50S subunit and demonstrate successful reconstitution of a functionally active 50S particle that can perform protein synthesis in vitro. We also successfully reconstituted both ribosomal subunits (30S and 50S) and 70S ribosomes using a full set of recombinant ribosomal proteins by integrating our developed method with the previously developed fully recombinant-based integrated synthesis, assembly and translation. The approach described here makes a major contribution to the field of ribosome engineering and could be fundamental to the future studies of ribosome assembly processes.

In vitro reconstitution of functional small ribosomal subunit assembly for comprehensive analysis of ribosomal elements in E. coli.

In vitro reconstitution is a powerful tool for investigating ribosome functions and biogenesis, as well as discovering new ribosomal features. In this study, we integrated all of the processes required for Escherichia coli small ribosomal subunit assembly. In our method, termed fully Recombinant-based integrated Synthesis, Assembly, and Translation (R-iSAT), assembly and evaluation of the small ribosomal subunits are coupled with ribosomal RNA (rRNA) synthesis in a reconstituted cell-free protein synthesis system. By changing the components of R-iSAT, including recombinant ribosomal protein composition, we coupled ribosomal assembly with ribosomal protein synthesis, enabling functional synthesis of ribosomal proteins and subsequent subunit assembly. In addition, we assembled and evaluated subunits with mutations in both rRNA and ribosomal proteins. The study demonstrated that our scheme provides new ways to comprehensively analyze any elements of the small ribosomal subunit, with the goal of improving our understanding of ribosomal biogenesis, function, and engineering.

Reconstituted cell-free protein synthesis using in vitro transcribed tRNAs.

Entire reconstitution of tRNAs for active protein production in a cell-free system brings flexibility into the genetic code engineering. It can also contribute to the field of cell-free synthetic biology, which aims to construct self-replicable artificial cells. Herein, we developed a system equipped only with in vitro transcribed tRNA (iVTtRNA) based on a reconstituted cell-free protein synthesis (PURE) system. The developed system, consisting of 21 iVTtRNAs without nucleotide modifications, is able to synthesize active proteins according to the redesigned genetic code. Manipulation of iVTtRNA composition in the system enabled genetic code rewriting. Introduction of modified nucleotides into specific iVTtRNAs demonstrated to be effective for both protein yield and decoding fidelity, where the production yield of DHFR reached about 40% of the reaction with native tRNA at 30°C. The developed system will prove useful for studying decoding processes, and may be employed in genetic code and protein engineering applications.

Live Single-Cell Mass Spectrometry (LSC-MS) for Plant Metabolomics.

Live single-cell mass spectrometry (LSC-MS) allows for the detection of hundreds to thousands of metabolite peaks acquired from a single plant cell within a few minutes. Plant cells are first observed under a stereomicroscope, a cell of interest is chosen, and then sampled using a metal-coated glass microcapillary for subsequent analysis. A few microliters of ionization solvent is then added to the rear end of the capillary followed by the introduction of the capillary's content directly into the mass spectrometer. High voltage is applied between the capillary and the mass spectrometer inlet to induce nanospray ionization. Metabolite structural confirmation is performed using tandem mass spectrometry analysis (MS/MS) and fragments are matched with MS/MS databases to predict metabolic pathways. This method enables swift and direct molecular detection and identification of specific metabolites from a single plant cell along with their localization within the cell, which will allow for comprehensive understanding of plant metabolomics on a single cell level.

Cell-free synthesis of stable isotope-labeled internal standards for targeted quantitative proteomics.

High-sensitivity mass spectrometry approaches using selected reaction monitoring (SRM) or multiple reaction monitoring (MRM) methods are powerful tools for targeted quantitative proteomics-based investigation of dynamics in specific biological systems. Both high-sensitivity detection of low-abundance proteins and their quantification using this technique employ stable isotope-labeled peptide internal standards. Currently, there are various ways for preparing standards, including chemical peptide synthesis, cellular protein expression, and cell-free protein or peptide synthesis. Cell-free protein synthesis (CFPS) or in vitro translation (IVT) systems in particular provide high-throughput and low-cost preparation methods, and various cell types and reconstituted forms are now commercially available. Herein, we review the use of such systems for precise and reliable protein quantification.

Peripheral Injection of Chicken Growth Hormone-Releasing Hormone Inhibits Feeding Behavior in Chicks.

Growth hormone-releasing hormone (GHRH), a stimulator of growth hormone (GH) secretion, is known to have several physiological roles such as the regulation of feeding behavior in mammals. Recently, we have reported that central injection of chicken GHRH decreased food intake in chicks, however, its peripheral role on feeding behavior has not been clarified. The purpose of the present study was to investigate the effect of peripheral injection of GHRH on feeding behavior in chicks (Gallus gallus). Intraperitoneal (IP) injection of GHRH47 (1 nmol), full length form of chicken GHRH significantly decreased food intake in chicks although the injection of GHRH27 and GHRH27-NH2, short forms of chicken GHRH had no effect. The IP injection of GHRH47 did not induced any abnormal behavior, suggesting that GHRH47-induced anorexia might not be related to abnormal behavior such as sleeping, hyperactivity and convulsion. The anorexigenic effect of GHRH47 seemed not to be related to GH because IP injection of bovine GH did not affect feeding behavior in chicks. Collectively, these results suggest that peripheral GHRH is related to inhibit feeding behavior in chicks.

Production and characterization of neurosecretory protein GM using Escherichia coli and Chinese Hamster Ovary cells.

Neurosecretory protein GL (NPGL) and neurosecretory protein GM (NPGM) are paralogs recently discovered in birds and in mammals. The post-translational products of NPGL and of NPGM genes include a signal peptide sequence, a glycine amidation signal, and a dibasic amino acid cleavage site. This suggests that the mature forms of NPGL and of NPGM are small proteins secreted in the hypothalamus and containing an amidated C-terminus. However, endogenous NPGL and NPGM have not yet been identified. Chicken NPGL and NPGM have two highly conserved Cys residues that are likely to form a disulfide bond, while mammalian NPGM has one additional Cys residue located between the two conserved Cys residues and the correct disulfide bond pattern is unclear. In this study, we prepared rat NPGM to elucidate the structure of its mature form. We first expressed the predicted mature NPGM, containing an extra C-terminal Gly, in Escherichia coli SHuffle cells, which are engineered to promote the formation of native disulfide bridges in recombinant proteins. We observed the presence of a disulfide bond between the N-terminal Cys residue and the second Cys residue, while the C-terminal Cys residue was free. Secondly, we transfected a construct containing the entire NPGM open reading frame into Chinese Hamster Ovary cells, and observed that NPGM was cleaved immediately after the signal peptide and that it was secreted into the medium. Furthermore, the protein presented a disulfide bond at the same location observed in recombinant NPGM.

Central administration of chicken growth hormone-releasing hormone decreases food intake in chicks.

Growth hormone-releasing hormone (GHRH) is well known as a stimulator of growth hormone (GH) secretion. GHRH not only stimulates GH release but also modifies feeding behavior and energy homeostasis in rodents. In chickens (Gallus gallus domesticus), on the other hand, two types of GHRH, namely, chicken GHRH (cGHRH) and cGHRH-like peptide (cGHRH-LP), have been identified. The purpose of the present study was to investigate the effect of central injection of cGHRH and cGHRH-LP on feeding behavior in chicks. Intracerebroventricular (ICV) injection of both cGHRH and cGHRH-LP (0.04 to 1 nmol) significantly decreased food intake without any abnormal behavior in chicks. Furthermore, the feeding-inhibitory effect was not abolished by co-injection of the antagonist for pituitary adenylate cyclase-activating polypeptide (PACAP) or corticotropin-releasing hormone (CRH) receptors, suggesting that the anorexigenic effect of cGHRH and cGHRH-LP might not be related to the PACAP and CRH systems in the brain of chicks. Finally, 24-h food deprivation increased mRNA expression of cGHRH but not cGHRH-LP in the diencephalon. These results suggest that central cGHRH is related to inhibiting feeding behavior and energy homeostasis in chicks.

Characterization of an avian histidine decarboxylase and localization of histaminergic neurons in the chicken brain.

In mammals, it is established that histamine is a neurotransmitter and/or neuromodulator in the central nervous system. It is produced by the enzyme histidine decarboxylase (HDC) in the tuberomammillary nucleus of the posterior hypothalamus. However, HDC as well as histaminergic neurons have not yet been characterized in the avian brain. We have cloned the cDNA for HDC from the chicken hypothalamus and demonstrated that the chicken HDC sequence is highly homologous to the mammalian counterpart, and that the expressed protein shows high enzymatic activity. The expression of HDC mRNA at various sites in the brain was investigated using quantitative RT-PCR. The results showed that the HDC mRNA was highly expressed in the hypothalamic infundibulum. In situ hybridization analyses revealed that the cells containing HDC mRNA were localized in the medial mammillary nucleus of the hypothalamic infundibulum. Intracerebroventricular injection of histamine in chicks resulted in inhibition of feeding behavior. This is the first report of the characterization of histaminergic neurons in the avian brain, and our findings indicate that neuronal histamine exerts anorexigenic effects in chicks.