12 research outputs found
Larval pufferfish protected by maternal tetrodotoxin
Marine pufferfish contain tetrodotoxin (TTX), an extremely potent neurotoxin. All species of the genus Takifugu accumulate TTX in the liver and ovaries, although the tissue(s) in which it is localized can differ among species. TTX is the major defense strategy the pufferfish appears to use against predators. TTX is also used as a male-attracting pheromone during spawning. Here we demonstrate an additional (and unexpected) use of maternal TTX in the early larval stages of the Takifugu pufferfish. Predation experiments demonstrated that juveniles of all the species of fish used as predators ingested pufferfish larvae, but spat them out promptly. Liquid Chromatography-Tandem Mass Spectrometry (LC-MSMS) analysis revealed that the pufferfish larvae contain a small quantity of TTX, which is not enough to be lethal to the predators. Immunohistochemical analysis with anti-TTX monoclonal antibody revealed that the TTX is primarily localized in the body surface of the larvae as a layer of protection. Our study showed the female parent of the Takifugu pufferfish vertically transfers TTX to the larvae through its accumulation in the ovaries, and subsequent localization on the body surface of the larvae
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. Keywords: Absolute quantification, Mass spectrometry, Cell-free protein synthesis system, In vitro translation, Targeted quantitative proteomics, PURE syste
Comparative Study of Cardiovascular Effects of Selected Pulmonary Vasodilators in Canine Models of Mitral Valve Disease
Previous reports have shown that various oral pulmonary vasodilators are effective against canine pulmonary hypertension (PH). However, no studies have compared their hemodynamic effects. We aimed to compare the hemodynamic effects of 15 µg/kg beraprost sodium, 1.0 mg/kg sildenafil, and their combination, in dogs with experimentally induced mitral regurgitation. This experimental crossover study evaluated the hemodynamic and functional effects of oral pulmonary vasodilators by application of right-sided heart catheterization and echocardiography. Beraprost significantly decreased pulmonary and systemic vascular resistance. Additionally, beraprost increased right-ventricular stroke volume and left-ventricular cardiac output without worsening left-heart size and left-atrial pressure. The pulmonary vasodilatory effects of sildenafil were stronger, and its systemic vasodilatory effects were weaker than those of beraprost. However, sildenafil significantly increased the left-ventricular volume, left-atrial pressure indicator, and right-ventricular cardiac output. Combination therapy resulted in the strongest pulmonary and systemic vasodilating effects without worsening the left-heart size and left-atrial pressure indicators. Both beraprost and sildenafil were effective against canine PH; however, sildenafil was associated with the risk of worsening left-heart loading. Combination therapy with beraprost and sildenafil synergistically dilated pulmonary and systemic vessels, indicating a more potent treatment option for severe PH cases
Mammalian reverse genetics without crossing reveals Nr3a as a short-sleeper gene
The identification of molecular networks at the system level in mammals is accelerated by next-generation mammalian genetics without crossing, which requires both the efficient production of whole-body biallelic knockout (KO) mice in a single generation and high-performance phenotype analyses. Here, we show that the triple targeting of a single gene using the CRISPR/Cas9 system achieves almost perfect KO efficiency (96%–100%). In addition, we developed a respiration-based fully automated noninvasive sleep phenotyping system, the Snappy Sleep Stager (SSS), for high-performance (95.3% accuracy) sleep/wake staging. Using the triple-target CRISPR and SSS in tandem, we reliably obtained sleep/wake phenotypes, even in double-KO mice. By using this system to comprehensively analyze all of the N-methyl-D-aspartate (NMDA) receptor family members, we found Nr3a as a short-sleeper gene, which is verified by an independent set of triple-target CRISPR. These results demonstrate the application of mammalian reverse genetics without crossing to organism-level systems biology in sleep research
Integration of pharmacoproteomic and computational approaches reveals the cellular signal transduction pathways affected by apatinib in gastric cancer cell lines
Apatinib is known to be a highly selective vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor with anti-angiogenic and anti-tumor properties. In a phase III study, the objective response rate to apatinib was low. It remains unclear why the effectivity of apatinib varies among patients and what type of patients are candidates for the treatment. In this study, we investigated the anti-tumor efficacy of apatinib against 13 gastric cancer cell lines and found that it differed depending on the cell line. Using integrated wet and dry approaches, we showed that apatinib was a multi-kinase inhibitor of c-Kit, RAF1, VEGFR1, VEGFR2, and VEGFR3, predominantly inhibiting c-Kit. Notably, KATO-III, which was the most apatinib-sensitive among the gastric cancer cell lines investigated, was the only cell line expressing c-Kit, RAF1, VEGFR1, and VEGFR3 but not VEGFR2. Furthermore, we identified SNW1 as a molecule affected by apatinib that plays an important role in cell survival. Finally, we identified the molecular network related to SNW1 that was affected by treatment with apatinib. These results suggest that the mechanism of action of apatinib in KATO-III cells is independent of VEGFR2 and that the differential efficacy of apatinib was due to differences in expression patterns of receptor tyrosine kinases. Furthermore, our results suggest that the differential efficacy of apatinib in gastric cell lines may be attributed to SNW1 phosphorylation levels at a steady state. These findings contribute to a deeper understanding of the mechanism of action of apatinib in gastric cancer cells
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Proteomic analysis of serum extracellular vesicles reveals Fibulin-3 as a new marker predicting liver-related events in MASLD.
BACKGROUND: There is a need for novel noninvasive markers for metabolic dysfunction-associated steatotic liver disease (MASLD) to stratify patients at high risk for liver-related events including liver cancer and decompensation. In the present study, we used proteomic analysis of proteins in extracellular vesicles (EVs) to identify new biomarkers that change with fibrosis progression and can predict the development of liver-related events. METHODS: We analyzed serum EVs from 50 patients with MASLD assessed for liver fibrosis by biopsy and identified proteins that altered with advanced fibrosis. A further evaluation was conducted on another cohort of 463 patients with MASLD with biopsy. RESULTS: Eight candidate proteins were identified by proteomic analysis of serum EVs. Among them, serum levels of Fibulin-3, Fibulin-1, and Ficolin 1 correlated with their EV levels. In addition, serum Fibulin-3 and serum Fibulin-1 levels changed significantly with advanced fibrosis. Using another cohort with biopsy, we found that the serum Fibulin-3 concentration was significantly greater in those with advanced fibrosis but that the serum Fibulin-1 concentration was not significantly different. Multivariate Cox proportional hazards analysis revealed that a higher Fibrosis-4 (FIB-4) index and higher serum Fibulin-3 concentration were independent risk factors for liver-related events. When the cutoff value for the serum Fibulin-3 concentration was 6.0 µg/mL according to the Youden index of AUROCs, patients with high serum Fibulin-3 significantly more frequently developed liver-related events than did other patients. Validation using another cohort of 226 patients with clinically diagnosed MASLD confirmed that high serum Fibulin-3 levels are associated with a greater frequency of liver-related events. CONCLUSIONS: Serum Fibulin-3 was identified as a biomarker for predicting liver-related events in patients with MASLD
A Strategy for Large-Scale Phosphoproteomics and SRM-Based Validation of Human Breast Cancer Tissue Samples
Protein phosphorylation is a key mechanism of cellular
signaling pathways and aberrant phosphorylation has been implicated
in a number of human diseases. Thus, approaches in phosphoproteomics
can contribute to the identification of key biomarkers to assess disease
pathogenesis and drug targets. Moreover, careful validation of large-scale
phosphoproteome analysis, which is lacking in the current protein-based
biomarker discovery, significantly increases the value of identified
biomarkers. Here, we performed large-scale differential phosphoproteome
analysis using IMAC coupled with the isobaric tag for relative quantification
(iTRAQ) technique and subsequent validation by selected/multiple reaction
monitoring (SRM/MRM) of human breast cancer tissues in high- and low-risk
recurrence groups. We identified 8309 phosphorylation sites on 3401
proteins, of which 3766 phosphopeptides (1927 phosphoproteins) were
able to be quantified and 133 phosphopeptides (117 phosphoproteins)
were differentially expressed between the two groups. Among them,
19 phosphopeptides were selected for further verification and 15 were
successfully quantified by SRM using stable isotope peptides as a
reference. The ratio of phosphopeptides between high- and low-risk
groups quantified by SRM was well correlated with iTRAQ-based quantification
with a few exceptions. These results suggest that large-scale phosphoproteome
quantification coupled with SRM-based validation is a powerful tool
for biomarker discovery using clinical samples
A Strategy for Large-Scale Phosphoproteomics and SRM-Based Validation of Human Breast Cancer Tissue Samples
Protein phosphorylation is a key mechanism of cellular
signaling pathways and aberrant phosphorylation has been implicated
in a number of human diseases. Thus, approaches in phosphoproteomics
can contribute to the identification of key biomarkers to assess disease
pathogenesis and drug targets. Moreover, careful validation of large-scale
phosphoproteome analysis, which is lacking in the current protein-based
biomarker discovery, significantly increases the value of identified
biomarkers. Here, we performed large-scale differential phosphoproteome
analysis using IMAC coupled with the isobaric tag for relative quantification
(iTRAQ) technique and subsequent validation by selected/multiple reaction
monitoring (SRM/MRM) of human breast cancer tissues in high- and low-risk
recurrence groups. We identified 8309 phosphorylation sites on 3401
proteins, of which 3766 phosphopeptides (1927 phosphoproteins) were
able to be quantified and 133 phosphopeptides (117 phosphoproteins)
were differentially expressed between the two groups. Among them,
19 phosphopeptides were selected for further verification and 15 were
successfully quantified by SRM using stable isotope peptides as a
reference. The ratio of phosphopeptides between high- and low-risk
groups quantified by SRM was well correlated with iTRAQ-based quantification
with a few exceptions. These results suggest that large-scale phosphoproteome
quantification coupled with SRM-based validation is a powerful tool
for biomarker discovery using clinical samples
A Strategy for Large-Scale Phosphoproteomics and SRM-Based Validation of Human Breast Cancer Tissue Samples
Protein phosphorylation is a key mechanism of cellular
signaling pathways and aberrant phosphorylation has been implicated
in a number of human diseases. Thus, approaches in phosphoproteomics
can contribute to the identification of key biomarkers to assess disease
pathogenesis and drug targets. Moreover, careful validation of large-scale
phosphoproteome analysis, which is lacking in the current protein-based
biomarker discovery, significantly increases the value of identified
biomarkers. Here, we performed large-scale differential phosphoproteome
analysis using IMAC coupled with the isobaric tag for relative quantification
(iTRAQ) technique and subsequent validation by selected/multiple reaction
monitoring (SRM/MRM) of human breast cancer tissues in high- and low-risk
recurrence groups. We identified 8309 phosphorylation sites on 3401
proteins, of which 3766 phosphopeptides (1927 phosphoproteins) were
able to be quantified and 133 phosphopeptides (117 phosphoproteins)
were differentially expressed between the two groups. Among them,
19 phosphopeptides were selected for further verification and 15 were
successfully quantified by SRM using stable isotope peptides as a
reference. The ratio of phosphopeptides between high- and low-risk
groups quantified by SRM was well correlated with iTRAQ-based quantification
with a few exceptions. These results suggest that large-scale phosphoproteome
quantification coupled with SRM-based validation is a powerful tool
for biomarker discovery using clinical samples