Hypoxia stabilizes SETDB1 to maintain genome stability.

Von Hippel-Lindau (VHL) is a tumor suppressor that functions as the substrate recognition subunit of the CRL2VHL E3 complex. While substrates of VHL have been identified, its tumor suppressive role remains to be fully understood. For further determination of VHL substrates, we analyzed the physical interactome of VHL and identified the histone H3K9 methyltransferase SETBD1 as a novel target. SETDB1 undergoes oxygen-dependent hydroxylation by prolyl hydroxylase domain proteins and the CRL2VHL complex recognizes hydroxylated SETDB1 for ubiquitin-mediated degradation. Under hypoxic conditions, SETDB1 accumulates by escaping CRL2VHL activity. Loss of SETDB1 in hypoxia compared with that in normoxia escalates the production of transposable element-derived double-stranded RNAs, thereby hyperactivating the immune-inflammatory response. In addition, strong derepression of TEs in hypoxic cells lacking SETDB1 triggers DNA damage-induced death. Our collective results support a molecular mechanism of oxygen-dependent SETDB1 degradation by the CRL2VHL E3 complex and reveal a role of SETDB1 in genome stability under hypoxia.

Structure-activity relationship analysis of novel GSPT1 degraders based on benzotriazinone scaffold and its antitumor effect on xenograft mouse model.

Molecular glue degraders, such as lenalidomide and pomalidomide, bind to cereblon (CRBN) E3 ligase and subsequently recruit neosubstrate proteins, Ikaros (IKZF1) and Aiolos (IKZF3), for the ubiquitination-proteasomal degradation process. In this study, we explored structure-activity relationship analysis for novel GSPT1 degraders utilizing a benzotriazinone scaffold previously discovered as a novel CRBN binder. In particular, we focused on the position of the ureido group on the benzotriazinone scaffold, substituent effect on the phenylureido group, and methyl substitution on the benzylic position of benzotriazinone. As a result, we identified 34f (TD-522), which exhibits strong anti-proliferative effects in both KG-1 (EC50 = 0.5 nM) and TMD-8 (EC50 = 5.2 nM) cell lines. Compound 34f effectively induced GSPT1 degradation with a DC50 of 0.269 nM and Dmax of >95 % at 10 nM concentration in KG-1 cells. An in vivo xenograft study showed that compound 34f effectively suppressed TMD8-driven tumor growth, suggesting a potential role in the development of novel GSPT1 degraders.

Chrysin-Induced G Protein-Coupled Estrogen Receptor Activation Suppresses Pancreatic Cancer.

Pancreatic cancer (PC) has a high mortality rate due to its poor prognosis and the possibility of surgical resection in patients with the disease. Importantly, adjuvant chemotherapy is necessary to improve PC prognosis. Chrysin, a natural product with anti-inflammatory, antioxidant, and anticancer properties, has been studied for several years. Our previous study demonstrated that chrysin induced G protein-coupled estrogen receptor (GPER) expression and regulated its activity in breast cancer. Herein, we investigated whether chrysin-induced GPER activation suppresses PC progression in MIA PaCa-2 cells and a xenograft model. To determine its mechanism of action, cytotoxicity and clonogenic assays, a FACS analysis, and Western blotting were performed. Furthermore, the delay in tumor growth was evaluated in the MIA PaCa-2-derived xenograft model. Tumor tissues were investigated by Western blotting, immunohistochemistry, and a proteomic analysis. Chrysin caused cell cycle arrest and significantly decreased cell viability. Following co-treatment with chrysin and 17ß-estradiol, the inhibitory effect of chrysin on cell proliferation was enhanced. In the xenograft model, chrysin and G1 (a GPER agonist) significantly delayed tumor growth and reduced both Ki-67 (a proliferation marker) and c-Myc expressions in tumor tissues. The proteomic analysis of tumor tissues identified that rho-associated coiled-coil containing protein kinase 1 (ROCK1), transgelin 2 (TAGLN2), and FCH and Mu domain containing endocytic adaptor 2 (FCHO2) levels were significantly reduced in chrysin-treated tumor tissues. High ROCK1, TAGLN2, and FCHO2 expressions were indicative of low overall PC survival as found using the Kaplan-Meier plotter. In conclusion, our results suggest that chrysin suppresses PC progression through the activation of GPER and reductions in ROCK1, TAGLN2, and FCHO2 expressions.

Covalent binding of uracil DNA glycosylase UdgX to abasic DNA upon uracil excision.

Uracil DNA glycosylases (UDGs) are important DNA repair enzymes that excise uracil from DNA, yielding an abasic site. Recently, UdgX, an unconventional UDG with extremely tight binding to DNA containing uracil, was discovered. The structure of UdgX from Mycobacterium smegmatis in complex with DNA shows an overall similarity to that of family 4 UDGs except for a protruding loop at the entrance of the uracil-binding pocket. Surprisingly, H109 in the loop was found to make a covalent bond to the abasic site to form a stable intermediate, while the excised uracil remained in the pocket of the active site. H109 functions as a nucleophile to attack the oxocarbenium ion, substituting for the catalytic water molecule found in other UDGs. To our knowledge, this change from a catalytic water attack to a direct nucleophilic attack by the histidine residue is unprecedented. UdgX utilizes a unique mechanism of protecting cytotoxic abasic sites from exposure to the cellular environment.

Molecular-Level Understanding of Continuous Growth from Iron-Oxo Clusters to Iron Oxide Nanoparticles.

The formation of inorganic nanoparticles has been understood based on the classical crystallization theory described by a burst of nucleation, where surface energy is known to play a critical role, and a diffusion-controlled growth process. However, this nucleation and growth model may not be universally applicable to the entire nanoparticle systems because different precursors and surface ligands are used during their synthesis. Their intrinsic chemical reactivity can lead to a formation pathway that deviates from a classical nucleation and growth model. The formation of metal oxide nanoparticles is one such case because of several distinct chemical aspects during their synthesis. Typical carboxylate surface ligands, which are often employed in the synthesis of oxide nanoparticles, tend to continuously remain on the surface of the nanoparticles throughout the growth process. They can also act as an oxygen source during the growth of metal oxide nanoparticles. Carboxylates are prone to chemical reactions with different chemical species in the synthesis such as alcohol or amine. Such reactions can frequently leave reactive hydroxyl groups on the surface. Herein, we track the entire growth process of iron oxide nanoparticles synthesized from conventional iron precursors, iron-oleate complexes, with strongly chelating carboxylate moieties. Mass spectrometry studies reveal that the iron-oleate precursor is a cluster comprising a tri-iron-oxo core and carboxylate ligands rather than a mononuclear complex. A combinatorial analysis shows that the entire growth, regulated by organic reactions of chelating ligands, is continuous without a discrete nucleation step.

Structural and mechanistic insights into the inhibition of class C ß-lactamases through the adenylylation of the nucleophilic serine.

Objectives: : Investigation into the adenylylation of the nucleophilic serine in AmpC BER and CMY-10 extended-spectrum class C ß-lactamases. Methods: : The formation and the stability of the adenylate adduct were examined by X-ray crystallography and MS. Inhibition assays for kinetic parameters were performed by monitoring the hydrolytic activity of AmpC BER and CMY-10 using nitrocefin as a reporter substrate. The effect of adenosine 5'-(P-acetyl)monophosphate (acAMP) on the MIC of ceftazidime was tested with four Gram-negative clinical isolates. Results: : The crystal structures and MS analyses confirmed the acAMP-mediated adenylylation of the nucleophilic serine in AmpC BER and CMY-10. acAMP inhibited AmpC BER and CMY-10 through the adenylylation of the nucleophilic serine, which could be modelled as a two-step mechanism. The initial non-covalent binding of acAMP to the active site is followed by the covalent attachment of its AMP moiety to the nucleophilic serine. The inhibition efficiencies ( k inact / K I ) of acAMP against AmpC BER and CMY-10 were determined to be 320 and 140 M -1 s -1 , respectively. The combination of ceftazidime and acAMP reduced the MIC of ceftazidime against the tested bacteria. Conclusions: : Our structural and kinetic studies revealed the detailed mechanism of adenylylation of the nucleophilic serine and may serve as a starting point for the design of novel class C ß-lactamase inhibitors on the basis of the nucleotide scaffold.

Covalent and Oriented Surface Immobilization of Antibody Using Photoactivatable Antibody Fc-Binding Protein Expressed in Escherichia coli.

Fc-specific antibody binding proteins (FcBPs) with the minimal domain of protein G are widely used for immobilization of well-oriented antibodies onto solid surfaces, but the noncovalently bound antibodies to FcBPs are unstable in sera containing large amounts of antibodies. Here we report novel photoactivatable FcBPs with photomethionine (pMet) expressed in E. coli, which induce Fc-specific photo-cross-linking with antibodies upon UV irradiation. Unfortunately, pMet did not support protein expression in the native E. coli system, and therefore we also developed an engineered methionyl tRNA synthetase (MRS5m). Coexpression of MRS5m proteins successfully induced photoactivatable FcBP overexpression in methionine-auxotroph E. coli cells. The photoactivatable FcBPs could be easily immobilized on beads and slides via their N-terminal cysteine residues and 6xHis tag. The antibodies photo-cross-linked onto the photoactivatable FcBP-beads were resistant from serum-antibody mediated dissociation and efficiently captured antigens in human sera. Furthermore, photo-cross-linked antibody arrays prepared using this system allowed sensitive detection of antigens in human sera by sandwich immunoassay. The photoactivatable FcBPs will be widely applicable for well-oriented antibody immobilization on various surfaces of microfluidic chips, glass slides, and nanobeads, which are required for development of sensitive immunosensors.

Formation of gas-phase peptide ions and their dissociation in MALDI: insights from kinetic and ion yield studies.

Insights on mechanisms for the generation of gas-phase peptide ions and their dissociation in matrix-assisted laser desorption ionization (MALDI) gained from the kinetic and ion yield studies are presented. Even though the time-resolved photodissociation technique was initially used to determine the dissociation kinetics of peptide ions and their effective temperature, it was replaced by a simpler method utilizing dissociation yields from in-source decay (ISD) and post-source decay (PSD). The ion yields for a matrix and a peptide were measured by repeatedly irradiating a region on a sample and collecting ion signals until the sample in the region was completely depleted. Matrix- and peptide-derived gas-phase cations were found to be generated by pre-formed ion emission or by ion-pair emission followed by anion loss, but not by laser-induced ionization. The total number of ions, that is, matrix plus peptide, was found to be equal to the number of ions emitted from a pure matrix. A matrix plume was found to cool as it expanded, from around 800-1,000 K to 400-500 K. Dissociation of peptide ions along b/y channels was found to occur statistically, that is, following RRKM behavior. Small critical energy (E0 = 0.6-0.7 eV) and highly negative critical entropy (ΔS(‡) = -30 to -25 eu) suggested that the transition structure was stabilized by multiple intramolecular interactions.

Discovery of a solvent effect preventing quantitative profiling by matrix-assisted laser desorption/ionization and its treatment.

RATIONALE: In analyte profiling by matrix-assisted laser desorption/ionization (MALDI), drawing a quantitative profile map is an outstanding problem. Recently, we developed a method to quantify an analyte by MALDI, which is needed to solve the problem. Another requirement for quantitative profiling is the quantitative sample-to-matrix analyte transfer, which is investigated in this work. METHODS: MALDI-time-of-flight (TOF) spectra were acquired for samples produced by two methods. In one, a sample solution containing a matrix and an analyte was loaded with a pipet and dried. In the other, a sample was prepared by a consecutive process, i.e., loading-drying of an analyte solution followed by that of a matrix solution. Two different micro-spotters were used in the second method. Various mixtures of organic solvents with water were used to prepare matrix solutions. RESULTS: The organic solvent, matrix, and analyte used in the study did not affect the analyte transfer efficiency, whereas it improved as the water content in the solvent increased. It also improved as the liquid droplet emitted by a micro-spotter got larger. Use of a more polar solvent or a larger droplet increases the contact time between a solution droplet and the sample surface, which seems to be responsible for the improvement in the transfer efficiency. CONCLUSIONS: Sample-to-matrix analyte transfer occurred efficiently when polar solvents and/or large liquid droplets were used to produce solid samples for MALDI profiling with a micro-spotter. A long contact time between the sample surface and a matrix solution droplet is one of the requirements for quantitative profiling. Copyright © 2015 John Wiley & Sons, Ltd.

A comparison of biochemical pregnancy rates between women who underwent IVF and fertile controls who conceived spontaneously†.

STUDY QUESTION: Does IVF affect the biochemical pregnancy rate? SUMMARY ANSWER: The likelihood of an early pregnancy loss may be lower and is certainly not higher in IVF cycles when compared with published rates of biochemical pregnancy in fertile women ≤42 years old. WHAT IS KNOWN ALREADY: The use of gonadotrophins to stimulate multi-folliculogenesis alters endometrial expression of genes and proteins, compared with unstimulated cycles. Exogenous estrogen and progesterone taken for endometrial preparation in frozen embryo transfer cycles, also cause changes in endometrial gene and protein expression .These endometrial alterations may compromise the ability of embryos to develop once implanted, possibly increasing the biochemical pregnancy rate. STUDY DESIGN, SIZE, DURATION: This is a retrospective study, involving 1636 fresh and 188 frozen, single embryo transfer (SET) IVF cycles performed between August 2008 and December 2012. The biochemical pregnancy rate of the 1824 combined IVF and frozen cycles were compared with fertile controls, derived from the three prospective studies in the medical literature that evaluate this rate. PARTICIPANTS/MATERIALS, SETTING, METHODS: Subjects ≤42-years old, who underwent a SET, as part of a fresh or thawed IVF cycle were considered for inclusion. Each subject is represented only once. The biochemical pregnancy rates were compared with those of historical standard, fertile populations with spontaneous conceptions. MAIN RESULTS AND THE ROLE OF CHANCE: The pregnancy rates per transfer for fresh and frozen IVF cycles were similar at 39 and 40%, respectively. There was also no significant difference in the likelihood of pregnancy outcomes (clinical, biochemical and ectopic pregnancy) between fresh IVF and frozen cycles (85.4 versus 85.6%, 13.8 versus 14.8%, 0.5 versus 0%, P = 0.82). However, pregnancy rates decreased in older patients when compared with younger ones P < 0.0001. The biochemical pregnancy rate for fresh and frozen IVF cycles combined was 13.8% of all pregnancies. IVF and frozen cycles were combined as the IVF group treated with hormones for further comparison with the fertile control group. The biochemical pregnancy rate (14%) in the IVF group was lower than the rate based on the total fertile group (18%), P = 0.01 and differed significantly from the rate in two out of the three studies used to establish the normative rate. The age ranges of the IVF and fertile controls were 21-42 years. The mean age in the IVF population was 34.8 years, as compared with 29 years, 29, 4 years and 30.6 years (Zinaman) in the three published studies (mean: 29.4 years). LIMITATIONS, REASONS FOR CAUTION: This is a retrospective study and it was impossible to recruit an in-house biochemical pregnancy control population. WIDER IMPLICATIONS OF THE FINDINGS: Lower early pregnancy wastage after IVF may be due to the opportunity to select the embryo for transfer. This finding should be confirmed in further studies but supports the idea that embryo selection is an important step. STUDY FUNDING/COMPETING INTERESTS: None.

Acquisition of the depth profiles and reproducible mass spectra in matrix-assisted laser desorption/ionization of inhomogeneous samples.

RATIONALE: In our previous analysis of the matrix-assisted laser desorption/ionization (MALDI) spectra of peptides, we treated their depth profiles in solid samples as homogeneous. Here, we wanted to determine if the reproducible MALDI spectra and linear calibration curves reported previously would be obtained even when the depth profiles were inhomogeneous. METHODS: We derived a formula relating shot-number-dependent ion abundance data in temperature-controlled MALDI with the analyte depth profile in a solid sample. We prepared samples containing peptides, amino acids, and serotonin in α-cyano-4-hydroxycinnamic acid matrix by vacuum-drying and micro-spotting methods, recorded their MALDI spectra, and analyzed them with the aforementioned formula. RESULTS: For the samples prepared by vacuum-drying, the analyte depth profiles were inhomogeneous and maximized at the sample surface. Although the MALDI spectra changed as the shot continued, their sum over the entire set of spectra acquired from a spot was reproducible. Similarly, a high-quality calibration curve could be obtained with the spectral data summed over the entire set. Depth profiles were homogeneous for samples prepared by micro-spotting. CONCLUSIONS: A method has been developed to obtain a reproducible MALDI spectrum and a linear calibration curve for an analyte with an inhomogeneous depth profile in a solid sample.

Dual track time-of-flight mass spectrometry for peptide quantification with matrix-assisted laser desorption/ionization.

RATIONALE: Previously, we reported a method (Anal. Chem. 2012, 84, 10332) for peptide quantification based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). In the method, the peptide-to-matrix ion abundance ratio was utilized. Implementation of the method with a commercial MALDI-TOF can be somewhat inconvenient because matrix-derived ions are routinely deflected away to avoid detector saturation. A solution for this inconvenience is required. METHODS: We installed a detector to acquire the TOF spectrum of the ions thrown away to avoid detector saturation. By sending the matrix- and peptide-derived ions along two different tracks and detecting them with different detectors, the inconvenience mentioned above could be avoided. RESULTS: Excellent linearity of the calibration curves obtained by the dual track TOF spectrometry is demonstrated. The method also allows for the acquisition of the tandem mass spectrum of a selected peptide, which can be useful for its identification. CONCLUSIONS: We devised the dual track MALDI-TOF MS method to avoid detector saturation and demonstrated that the quantification and identification of peptides can be performed simultaneously.

Angiotensin-converting enzyme inhibition prevents l-dopa-induced dyskinesia in a 6-ohda-induced mouse model of Parkinson's disease.

Parkinson's disease (PD) is characterised by severe movement defects and the degeneration of dopaminergic neurones in the midbrain. The symptoms of PD can be managed with dopamine replacement therapy using L-3, 4-dihydroxyphenylalanine (L-dopa), which is the gold standard therapy for PD. However, long-term treatment with L-dopa can lead to motor complications. The central renin-angiotensin system (RAS) is associated with the development of neurodegenerative diseases in the brain. However, the role of the RAS in dopamine replacement therapy for PD remains unclear. Here, we tested the co-treatment of the angiotensin-converting enzyme inhibitor (ACEI) with L-dopa altered L-dopa-induced dyskinesia (LID) in a 6-hydroxydopamine (6-OHDA)-lesioned mouse model of PD. Perindopril, captopril, and enalapril were used as ACEIs. The co-treatment of ACEI with L-dopa significantly decreased LID development in 6-OHDA-lesioned mice. In addition, the astrocyte and microglial transcripts involving Ccl2, C3, Cd44, and Iigp1 were reduced by co-treatment with ACEI and L-dopa in the 6-OHDA-lesioned striatum. In conclusion, co-treatment with ACEIs and L-dopa, such as perindopril, captopril, and enalapril, may mitigate the severity of L-DOPA-induced dyskinesia in a mouse model of PD.

Matrix suppression as a guideline for reliable quantification of peptides by matrix-assisted laser desorption ionization.

We propose to divide matrix suppression in matrix-assisted laser desorption ionization into two parts, normal and anomalous. In quantification of peptides, the normal effect can be accounted for by constructing the calibration curve in the form of peptide-to-matrix ion abundance ratio versus concentration. The anomalous effect forbids reliable quantification and is noticeable when matrix suppression is larger than 70%. With this 70% rule, matrix suppression becomes a guideline for reliable quantification, rather than a nuisance. A peptide in a complex mixture can be quantified even in the presence of large amounts of contaminants, as long as matrix suppression is below 70%. The theoretical basis for the quantification method using a peptide as an internal standard is presented together with its weaknesses. A systematic method to improve quantification of high concentration analytes has also been developed.

Spindle examination in unfertilized eggs using the polarization microscope can assist rescue ICSI.

Rescue ICSI can induce a high rate of 3 pronuclei (PN) formation from double insemination in eggs already fertilized by IVF but lacking signs of normal pronuclear formation. This study was performed to determine whether the number of 3PN embryos could be reduced by using the polarization microscope for rescue intracytoplasmic sperm injection (ICSI). As a study group, after conventional insemination, 81 unfertilized mature oocytes from 11 couples were checked for the number of spindles using the polarization microscope. One spindle (82.7%) or two spindles (17.3%) were observed in this group. Rescue ICSI was only performed on the unfertilized oocytes showing one spindle. In the control group, 87 mature oocytes which lacked visualization of any fertilization signs were selected for rescue ICSI and none of them underwent observation of the spindle. After rescue ICSI, the normal fertilization rate in the study group was significantly higher than in the control (68.7% versus 43.7%; P=0.0032). The rate of 3PN or 4PN embryos was significantly decreased in the study group with one spindle compared with the group without observation of the spindle (4.5% versus 26.4%; P=0.0004).

The developmental competence of human metaphase I oocytes with delayed maturation in vitro.

OBJECTIVE: To evaluate whether metaphase I (MI) oocytes completing maturation in vitro to metaphase II ("MI-MII oocytes") have similar developmental competence as the sibling metaphase II (MII) oocytes that reached maturity in vivo. DESIGN: Retrospective cohort study. SETTING: Academic medical center. PATIENT(S): A total of 1,124 intracytoplasmic sperm injection (ICSI) cycles from 800 patients at a single academic center between April 2016 and December 2020 with at least 1 MII oocyte immediately after retrieval and at least 1 sibling "MI-MII oocyte" that was retrieved as MI and matured to MII in culture before ICSI were included in the study. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): A total of 7,865 MII and 2,369 sibling MI-MII oocytes retrieved from the same individuals were compared for the fertilization and blastocyst formation rates. For patients who underwent single euploid blastocyst transfers (n = 406), the clinical pregnancy, spontaneous pregnancy loss, and live birth rates were compared between the 2 groups. RESULT(S): The fertilization rate was significantly higher in MII oocytes than in delayed matured MI-MII oocytes (75.9% vs. 56.1%). Similarly, the blastocyst formation rate was higher in embryos derived from MII oocytes than in those from MI-MII oocytes (53.8% vs. 23.9%). The percentage of euploid embryos derived from MII oocytes was significantly higher than that of those from MI-MII oocytes (49.2% vs. 34.7%). Paired comparison of sibling oocytes within the same cycle showed higher developmental competence of the MII oocytes than that of MI-MII oocytes. However, the pregnancy, spontaneous pregnancy loss, and live birth rates after a single euploid blastocyst transfer showed no statistically significant difference between the 2 groups (MII vs. MI-MII group, 65.7% vs. 74.1%, 6.4% vs. 5.0%, and 61.5% vs. 70.0%, respectively). CONCLUSION(S): Compared with oocytes that matured in vivo and were retrieved as MII, the oocytes that were retrieved as MI and matured to MII in vitro before ICSI showed lower developmental competence, including lower fertilization, blastocyst formation, and euploidy rates. However, euploid blastocysts from either cohort resulted in similar live birth rates, indicating that the MI oocytes with delayed maturation can still be useful even though the overall developmental competence was lower than that of their in vivo matured counterparts.

Investigation of Phospholipid Differences in Valproic Acid-Induced Autistic Mouse Model Brain Using Mass Spectrometry Imaging.

Autism is a neurodevelopmental disorder for which the cause and treatment have yet not been determined. The polyunsaturated fatty acid (PUFA) levels change rapidly in the blood or cerebrospinal fluid of autistic children and PUFAs are closely related to autism spectrum disorder (ASD). This finding suggests that changes in lipid metabolism are associated with ASD and result in an altered distribution of phospholipids in cell membranes. To further understand ASD, it is necessary to analyze phospholipids in organs consisting of nerve cells, such as the brain. In this study, we investigated the phospholipid distribution in the brain tissue of valproic acid-induced autistic mice using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Phospholipids including phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine were identified in each brain region and exhibited differences between the ASD and control groups. These phospholipids contain docosahexaenoic acid and arachidonic acid, which are important PUFAs for cell signaling and brain growth. We expect that the differences in phospholipids identified in the brain tissue of the ASD model with MALDI-MSI, in conjunction with conventional biological fluid analysis, will help to better understand changes in lipid metabolism in ASD.

Comparison study of mouse brain tissue by using ToF-SIMS within static limits and hybrid SIMS beyond static limits (dynamic mode).

In the study of degenerative brain diseases, changes in lipids, the main component of neurons, are particularly important because they are used as indicators of pathological changes. One method for the sensitive measurement of biomolecules, especially lipids, is time-of-flight secondary ion mass spectrometry (ToF-SIMS) using pulsed argon cluster ions. In this study, biomolecules including various lipids present in normal mouse brain tissue were measured using ToF-SIMS equipped with pulsed argon cluster primary ions. Based on the ToF-SIMS measurement results, hybrid SIMS (OrbiSIMS), which is a ToF-SIMS system with the addition of an orbitrap mass analyzer, was used to directly identify the biomolecules by the region in the real tissue samples. For this, the results of ToF-SIMS, which measured the tissue samples from a single mouse brain within static limits, were compared with those from OrbiSIMS measured beyond the static limits in terms of the differences in molecular profiling. From this analysis, two types of positive and negative ions were selected for identification, with the OrbiSIMS MS/MS results indicating that the positive ions were glycerophosphocholine and the negative ions were glycerophosphoinositol and sulfatide, a sphingolipid. Then, to confirm the identification of the molecular candidates, lipids were extracted from mirror image tissue samples, and LC-MS/MS also using an orbitrap mass analyzer was performed. As a result, the direct identification of molecular candidate groups distributed in particular regions of the tissue samples via OrbiSIMS was found to be consistent with the identification results by LC-MS/MS for extracted samples.

Phytochrome B photobodies are comprised of phytochrome B and its primary and secondary interacting proteins.

Phytochrome B (phyB) is a plant photoreceptor that forms a membraneless organelle called a photobody. However, its constituents are not fully known. Here, we isolated phyB photobodies from Arabidopsis leaves using fluorescence-activated particle sorting and analyzed their components. We found that a photobody comprises ~1,500 phyB dimers along with other proteins that could be classified into two groups: The first includes proteins that directly interact with phyB and localize to the photobody when expressed in protoplasts, while the second includes proteins that interact with the first group proteins and require co-expression of a first-group protein to localize to the photobody. As an example of the second group, TOPLESS interacts with PHOTOPERIODIC CONTROL OF HYPOCOTYL 1 (PCH1) and localizes to the photobody when co-expressed with PCH1. Together, our results support that phyB photobodies include not only phyB and its primary interacting proteins but also its secondary interacting proteins.

Evaluation of the effect of dimethyl fumarate on human bone marrow-derived mesenchymal stem cells using bottom-up proteomics.

Mesenchymal stem cells (MSCs) have potential as a viable treatment option in the field of regenerative medicine, but MSC-based therapy needs to be more efficient. Preconditioning is a method to improve MSC-based therapy, and dimethyl fumarate (DMF) - an agent that can enhance the antioxidative capacity of cells - can be considered for preconditioning of MSCs. In this study, we treated bone marrow-derived MSCs with DMF and evaluated their proteome using bottom-up proteomics. The MSCs were exposed to 10 µM DMF for 24 h, followed by lysis with an SDS solution, digestion with trypsin using an s-trap column, and analysis using nanoLC-MS/MS, which identified 2262 proteins with confidence. Bioinformatic analysis of the identified proteins revealed 47 upregulated proteins and 81 downregulated proteins upon DMF treatment. Pathway enrichment analysis suggested a possible decrease in autophagy and a decrease in the activity of the TCA cycle, while indicating a potential increase in proliferation and antioxidant activity in DMF-treated MSCs compared to untreated MSCs. Our findings suggest that DMF can enhance the proliferation of MSCs and increase their stability, and that preconditioning could improve the therapeutic efficacy of MSCs for the treatment of regenerative diseases.