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Suppression of starch branching enzymes 1 and 2 in cassava leads to increased resistant starch content through the production of high-amylose and modification of the amylopectin structure. Cassava (Manihot esculenta Crantz) is a starchy root crop used for human consumption as a staple food and industrial applications. Starch is synthesized by various isoforms of several enzymes. However, the function of starch branching enzymes (SBEs) in starch biosynthesis and mechanisms of starch regulation in cassava have not been understood well. In this study, we aimed to suppress the expression of SBEs in cassava to generate starches with a range of distinct properties, in addition to verifying the functional characteristics of the SBEs. One SBE1, two SBE2, and one SBE3 genes were classified by phylogenetic analysis and amino acid alignment. Quantitative real-time RT-PCR revealed tissue-specific expression of SBE genes in the tuberous roots and leaves of cassava. We introduced RNAi constructs containing fragments of SBE1, SBE2, or both genes into cassava by Agrobacterium-mediated transformation, and assessed enzymatic activity of SBE using tuberous roots and leaves from these transgenic plants. Simultaneous suppression of SBE1 and SBE2 rendered an extreme starch phenotype compared to suppression of SBE2 alone. Degree of polymerization of 6-13 chains in amylopectin was markedly reduced by suppression of both SBE1 and SBE2 in comparison to the SBE2 suppression; however, no change in chain-length profiles was observed in the SBE1 suppression alone. The role of SBE1 and SBE2 may have functional overlap in the storage tissue of cassava. Simultaneous suppression of SBE1 and SBE2 resulted in highly resistant starch with increased apparent amylose content compared to suppression of SBE2 alone. This study provides valuable information for understanding starch biosynthesis and suggests targets for altering starch quality.

RNA in extracellular vesicles (EVs) are uptaken by cells, where they regulate fundamental cellular functions. EV-derived mRNA in recipient cells can be translated. However, it is still elusive whether “naked nonvesicular extracellular mRNA” (nex-mRNA) that are not packed in EVs can be uptaken by cells and, if so, whether they have any functions in recipient cells. Here, we show the entrance of nex-mRNA in the nucleus, where they exert a translation-independent function. Human nex-interleukin-1β (IL1β)-mRNA outside cells proved to be captured by RNA-binding zinc finger CCCH domain containing protein 12D (ZC3H12D)-expressing human natural killer (NK) cells. ZC3H12D recruited to the cell membrane binds to the 3′-untranslated region of nex-IL1β-mRNA and transports it to the nucleus. The nex-IL1β-mRNA in the NK cell nucleus upregulates antiapoptotic gene expression, migration activity, and interferon-γ production, leading to the killing of cancer cells and antimetastasis in mice. These results implicate the diverse actions of mRNA., Nonvesicular extracellular RNA (nex-RNA) that are not packed in extracellular vesicles is detected outside the cell, but it is poorly understood. Here the authors report that nex-RNA is captured by a zinc finger protein and transported to the nucleus to enhance antimetastatic characters of the cell.

DNA binding proteins rapidly locate their specific DNA targets through a combination of 3D and 1D diffusion mechanisms, with the 1D search involving bidirectional sliding along DNA. However, even in nucleosome-free regions, chromosomes are highly decorated with associated proteins that may block sliding. Here we investigate the ability of the abundant chromatin-associated HMGB protein Nhp6A from Saccharomyces cerevisiae to travel along DNA in the presence of other architectural DNA binding proteins using single-molecule fluorescence microscopy. We observed that 1D diffusion by Nhp6A molecules is retarded by increasing densities of the bacterial proteins Fis and HU and by Nhp6A, indicating these structurally diverse proteins impede Nhp6A mobility on DNA. However, the average travel distances were larger than the average distances between neighboring proteins, implying Nhp6A is able to bypass each of these obstacles. Together with molecular dynamics simulations, our analyses suggest two binding modes: mobile molecules that can bypass barriers as they seek out DNA targets, and near stationary molecules that are associated with neighboring proteins or preferred DNA structures. The ability of mobile Nhp6A molecules to bypass different obstacles on DNA suggests they do not block 1D searches by other DNA binding proteins.

Staphylococcus aureus uses IsdG and IsdI to convert heme into a mixture of staphylobilin isomers, 15-oxo-β-bilirubin and 5-oxo-δ-bilirubin, formaldehyde, and iron. The highly ruffled heme found in ...

Integrative omics approaches revealed a crosstalk among phytohormones during tuberous root development in cassava. Tuberous root formation is a complex process consisting of phase changes as well as cell division and elongation for radial growth. We performed an integrated analysis to clarify the relationships among metabolites, phytohormones, and gene transcription during tuberous root formation in cassava (Manihot esculenta Crantz). We also confirmed the effects of the auxin (AUX), cytokinin (CK), abscisic acid (ABA), jasmonic acid (JA), gibberellin (GA), brassinosteroid (BR), salicylic acid, and indole-3-acetic acid conjugated with aspartic acid on tuberous root development. An integrated analysis of metabolites and gene expression indicated the expression levels of several genes encoding enzymes involved in starch biosynthesis and sucrose metabolism are up-regulated during tuberous root development, which is consistent with the accumulation of starch, sugar phosphates, and nucleotides. An integrated analysis of phytohormones and gene transcripts revealed a relationship among AUX signaling, CK signaling, and BR signaling, with AUX, CK, and BR inducing tuberous root development. In contrast, ABA and JA inhibited tuberous root development. These phenomena might represent the differences between stem tubers (e.g., potato) and root tubers (e.g., cassava). On the basis of these results, a phytohormonal regulatory model for tuberous root development was constructed. This model may be useful for future phytohormonal studies involving cassava.

Experimental and theoretical investigations of the self-assembly process of a Pd(II) coordination M6L4 square-based pyramid (SP) were conducted. It was found that the probable self-assembly pathway, in which the dimerization of M2L2 with two M leads to SP, expected from the connectivity of the building blocks is not a major self-assembly pathway to the M6L4 SP. Whether the M6L4 SP is assembled or M2L2 is trapped is determined by an inter- or intramolecular reaction in a chain-like M2L2X, where X is a leaving ligand. The kinetically trapped state where the M6L4 SP is produced from M2L2 beyond the Boltzmann distribution was realized by a concentration-induced process and was kept for at least 2 months at 298 K.

Despite the continuous discovery of host and guest proteins in membraneless organelles, complex host–guest interactions hinder the understanding of the molecular grammar governing liquid–liquid phase separation. In this study, we characterized the localization and dynamic properties of guest proteins in liquid droplets using single-molecule fluorescence microscopy. Eighteen guest proteins of different sizes, structures, and oligomeric states were examined in host p53 liquid droplets. Recruitment did not significantly depend on the structural properties of the guest proteins, but was moderately correlated with their length, total charge, and number of R and Y residues. In contrast, the diffusion of disordered guest proteins was comparable to that of host p53, whereas that of folded proteins varied widely. Molecular dynamics simulations suggest that folded proteins diffuse within the voids of the liquid droplet while interacting weakly with neighboring host proteins, whereas disordered proteins adapt their structures to form tight interactions with the host proteins. Our study provides insights into the key molecular principles of the localization and dynamics of guest proteins in liquid droplets.

Leukemic oncoproteins cause uncontrolled self-renewal of hematopoietic progenitors by aberrant gene activation, eventually causing leukemia. However, the molecular mechanism underlying aberrant gene activation remains elusive. Here, we showed that leukemic MLL fusion proteins associate with the HBO1 histone acetyltransferase (HAT) complex through their trithorax homology domain 2 (THD2) in various human cell lines. MLL proteins associated with the HBO1 complex through multiple contacts mediated mainly by the ING4/5 and PHF16 subunits in a chromatin-bound context where histone H3 lysine 4 tri-methylation marks were present. Of the many MLL fusions, MLL-ELL particularly depended on the THD2-mediated association with the HBO1 complex for leukemic transformation. The C-terminal portion of ELL provided a binding platform for multiple factors including AF4, EAF1, and p53. MLL-ELL activated gene expression in murine hematopoietic progenitors by loading an AF4/ENL/P-TEFb (AEP) complex onto the target promoters wherein the HBO1 complex promoted the association with AEP complex over EAF1 and p53. Moreover, the NUP98-HBO1 fusion protein exerted its oncogenic properties via interaction with MLL but not its intrinsic HAT activity. Thus, the interaction between the HBO1 complex and MLL is an important nexus in leukemic transformation, which may serve as a therapeutic target for drug development., 白血病を引き起こすタンパク質間相互作用の発見. 京都大学プレスリリース. 2021-08-31.

To achieve precise control of sulfonated polymer structures, a series of poly(p-phenylene)-based ionomers with well-controlled ion exchange capacities (IECs) were synthesised via a three-step technique: (1) preceding sulfonation of the monomer with a protecting group, (2) nickel(0) catalysed coupling polymerisation, and (3) cleavage of the protecting group of the polymers. 2,2-Dimethylpropyl-4-[4-(2,5-dichlorobenzoyl)phenoxy]benzenesulfonate (NS-DPBP) was synthesised as the preceding sulfonated monomer by treatment with chlorosulfuric acid and neopentyl alcohol. NS-DPBP was readily soluble in various organic solvents and stable during the nickel(0) catalysed coupling reaction. Sulfonated poly(4-phenoxybenzoyl-1,4-phenylene) (S-PPBP) homopolymer and seven types of random copolymers (S-PPBP-co-PPBP) with different IECs were obtained by varying the stoichiometry of NS-DPBP. The IECs and weight average molecular weights (Mws) of ionomers were in the range of 0.41–2.84 meq. g−1 and 143 000–465 000 g mol−1, respectively. The water uptake, proton conductivities, and water diffusion properties of ionomers exhibited a strong IEC dependence. Upon increasing the IEC of S-PPBP-co-PPBPs from 0.86 to 2.40 meq. g−1, the conductivities increased from 6.9 × 10−6 S cm−1 to 1.8 × 10−1 S cm−1 at 90% RH. S-PPBP and S-PPBP-co-PPBP (4 : 1) with IEC values >2.40 meq. g−1 exhibited fast water diffusion (1.6 × 10−11 to 8.0 × 10−10 m2 s−1), and were comparable to commercial perfluorosulfuric acid polymers. When fully hydrated, the maximum power density and the limiting current density of membrane electrode assemblies (MEAs) prepared with S-PPBP-co-PPBP (4 : 1) were 712 mW cm−2 and 1840 mA cm−2, respectively.

True understanding of dispersion interaction in solution remains elusive because of difficulty in the precise evaluation of its interaction energy. Here, the effect of substituents with different polarizability on dispersion interactions in water is discussed based on the thermodynamic parameters determined by isothermal titration calorimetry for the formation of discrete aggregates from gear-shaped amphiphiles (GSAs). The substituents with higher polarizability enthalpically more stabilize the nanocube, which is due to stronger dispersion interactions and to the hydrophobic effect. The differences in the thermodynamic parameters for the nanocubes from the GSAs with CH3 and CD3 groups are also discussed to lead to the conclusion that the H/D isotope effect on dispersion interactions is negligibly small, which is due to almost perfect entropy-enthalpy compensation between the two isotopomers. The thermodynamic parameters of molecules dispersed in water are influenced by the polarizability of their substituents. Here the formation of discrete aggregates from gear-shaped amphiphiles is determined by isothermal titration calorimetry, showing that substituents with higher polarizability stabilize the nanocubes enthalpically.

Kinetic control of molecular self-assembly remains difficult because of insufficient understanding of molecular self-assembly mechanisms. Here we report the formation of a metastable [Pd2L4]4+ cage structure composed of naphthalene-based ditopic ligands (L) and Pd(II) ions in very high yield (99%) under kinetic control by modulating the energy landscape. When self-assembly occurs with anionic guests in weakly cooordinating solvent then suitable intermedites and the metastable cage is formed. These conditions also prevent further transformation into the thermodynamically decomposed state. The cage formation pathways under kinetic control and the effect of the anions encapsulated on the self-assembly processes were investigated by QASAP (quantitative analysis of self-assembly process) and NASAP (numerical analysis of self-assembly process). It was found that the self-assembly with a preferred guest (BF4 -) proceeds through intermediates composed of no more components than the cage ([PdaLbXc]2a+ (a ≤ 2, b ≤ 4, X indicates a leaving ligand)) and that the final intramolecular cage-closure step is the rate-determining step. In contrast, a weaker guest (OTf-) causes the transient formation of intermediates composed of more components than the cage ([PdaLbXc]2a+ (a > 2, b > 4)), which are finally converted into the cage.

We report the first discovery of Lower Triassic Clarkina assemblages: four natural conodont assemblages from Lower Triassic pelagic black claystones of the North Kitakami Belt in northeastern Japan (Akkamori section). The fossils were obtained from the 2.5-m horizon level above the black claystone base, which is assigned to the end-Permian mass-extinction event. This horizon has been dated to the earliest Triassic (Griesbachian) by the occurrence of Hindeodus parvus, which is the index species for the base of the Triassic, in the same and subjacent horizons. These four fossil assemblages include a paired segminiplanate-formed P1 element, which was identified as the genus Clarkina, and have fully or partially preserved the original components of conodont elements. The most complete assemblage among them includes 15 distinctive elements, namely S0 and pairs of M, S1, S2, S3, S4, P1, and P2. It is noteworthy that these fossil assemblages preserve probable impressions of ‘eyes,’ which were replaced by aggregations of silicate, phosphate, and sulphide minerals. The occurrence of several sets of fossils that retain the original positioning of the conodonts' elemental apparatuses, as well as the original presence of soft tissue, may be attributed to the process by which the conodonts' bodies were transported to the deep seafloor, and by which the activity of agents of decomposition was inhibited in near-abiotic sediments under anoxic conditions in the pelagic deep sea during the earliest Triassic.

Separation of a homogeneous mixture of different components to reach an ordered out-of-equilibrium state in solution has attracted continuous attention. While this can be achieved using external chemical fuels or photo energy, an alternative energy source is heat. Here we realize a temperature-controlled cycle of transitions between ordered and disordered states based on a mixture of two kinds of building blocks that self-assemble into cubic structures (nanocubes). An almost statistical mixture of nanocubes (disordered state) is thermodynamically most stable at lower temperature (25 °C), while homoleptic assemblies composed of single components are preferentially produced at higher temperature (100 °C) followed by rapid cooling. The scrambling of the building blocks between the nanocubes takes place through the exchange of free building blocks dissociated from the nanocubes. Based on this mechanism, it is possible to accelerate, retard, and perfectly block the scrambling by the guest molecules encapsulated in the nanocubes., In this paper, the authors study the temperature-controlled dynamic behavior of a system of nanocubes self-assembled from two different building blocks. Non-intuitively, the disordered, equilibrium state (a mixture of heteroleptic cubes) and the ordered, out-of-equilibrium state (a mixture of homoleptic cubes) are cycled by heating and subsequent rapid cooling.

A supramolecular approach is effective to construct topologically complicated molecules with the aid of reversible bond formation. Although topologically complicated molecules have been synthesized for the past three decades, their formation mechanisms have rarely been discussed. Here we report the formation process of a tetranuclear interlocked palladium cage composed of two binuclear cages, which are quadruply interlocked with each other. In the main pathway, the binuclear cages are produced with binuclear partial cages. The ditopic ligand that does not bridge the two palladium(II) ions in the binuclear partial cage then threads into the binuclear cage to afford a tetranuclear partially interlocked cage, with partial conversion of the binuclear cage into the binuclear partial cage. The tetranuclear partially interlocked cage interlocks intramolecularly through repetitive cleavage and formation of Pd(II)–N coordination bonds mediated by a free pyridyl group, finally leading to the tetranuclear interlocked cage. Mechanically interlocked molecules are widely studied, but the pathways by which they assemble are less well understood. Here the formation process of a quadruply-locked Pd4L8 cage is studied by NMR and mass spectrometry, with intermediates inferred by kinetic analysis.

A 17-year-old girl was diagnosed with acute lymphoblastic leukemia (ALL). After the administration of high-dose methotrexate (MTX) or intrathecal MTX, the patient experienced transient hemiparesis and motor aphasia. Diffusion-weighted magnetic resonance imaging showed a high-intensity lesion in the bilateral centrum semiovale, and a vasospasm was detected in the proximal segment of bilateral A1 on magnetic resonance angiography. Edaravone was administered, and leucovorin rescue treatment was continued; eventually, the patient's neurological symptoms completely resolved. This finding suggested that vasospasm might be a mechanism underlying MTX-induced transient encephalopathy in adolescent and young adult patients with ALL.

We encountered a 30-year-old woman with remarkably elevated luteinizing hormone (LH) levels, as measured by electrochemiluminescent immunoassay (ECLIA), and no specific symptoms. We performed the following investigations: dilution linearity test, polyethylene glycol (PEG) precipitation test, immunoprecipitation test, protein G addition test, and high-performance liquid chromatography (HPLC) analysis. The linearity of patient's serum was similar to that of a standard LH preparation, and non-specific reactions were not observed. The recovery rate of LH shown by the PEG precipitation test, immunoprecipitation test, and protein G addition test was low. Moreover, an abnormal peak in HPLC was located at a slightly larger molecular weight position than that of IgG. These results showed the presence of macro-LH, LH, and anti-LH-IgG autoantibody complex and suggested that the clearance of LH from the blood was delayed due to IgG binding, and therefore, the LH value was falsely high. We should keep the possibility of macro-LH in mind in cases of unexpectedly high LH values.

Purpose: Five-aminolevulinic acid (5-ALA) is widely used as an intraoperative fluorescent probe for radical resection of high-grade glioma, and thus aids in extending progression-free survival of patients. However, there exist some cases where 5-ALA fails to fluoresce. In some other cases, it may undergo fluorescence quenching but cannot be orally readministered during surgery. This study aimed to develop a novel hydroxymethyl rhodamine green (HMRG)-based fluorescence labeling system that can be repeatedly administered as a topical spray during surgery for the detection of glioblastoma. Experimental Design: We performed a three-stage probe screening using tumor lysates and fresh tumor tissues with our probe library consisting of a variety of HMRG probes with different dipeptides. We then performed proteome and transcript expression analyses to detect candidate enzymes responsible for cleaving the probe. Moreover, in vitro and ex vivo studies using U87 glioblastoma cell line were conducted to validate the findings. Results: The probe screening identified proline-arginine–HMRG (PR-HMRG) as the optimal probe that distinguished tumors from peritumoral tissues. Proteome analysis identified calpain-1 (CAPN1) to be responsible for cleaving the probe. CAPN1 was highly expressed in tumor tissues which reacted to the PR-HMRG probe. Knockdown of this enzyme suppressed fluorescence intensity in U87 glioblastoma cells. In situ assay using a mouse U87 xenograft model demonstrated marked contrast of fluorescence with the probe between the tumor and peritumoral tissues. Conclusions: The novel fluorescent probe PR-HMRG is effective in detecting glioblastoma when applied topically. Further investigations are warranted to assess the efficacy and safety of its clinical use.

A fine-tuned activation and deactivation of proteases and their inhibitors are involved in the execution of the inflammatory response. The zymogen/proenzyme plasminogen is converted to the serine protease plasmin, a key fibrinolytic factor by plasminogen activators including tissue-type plasminogen activator (tPA). Plasmin is part of an intricate protease network controlling proteins of initial hemostasis/coagulation, fibrinolytic and complement system. Activation of these protease cascades is required to mount a proper inflammatory response. Although best known for its ability to dissolve clots and cleave fibrin, recent studies point to the importance of fibrin-independent functions of plasmin during acute inflammation and inflammation resolution. In this review, we provide an up-to-date overview of the current knowledge of the enzymatic and cytokine-like effects of tPA and describe the role of tPA and plasminogen receptors in the regulation of the inflammatory response with emphasis on the cytokine storm syndrome such as observed during coronavirus disease 2019 or macrophage activation syndrome. We discuss tPA as a modulator of Toll like receptor signaling, plasmin as an activator of NFkB signaling, and summarize recent studies on the role of plasminogen receptors as controllers of the macrophage conversion into the M2 type and as mediators of efferocytosis during inflammation resolution., Graphical abstract Unlabelled Image, Highlights • The fibrinolytic factor plasmin interconnects with the coagulation and complement system. • Plasmin modulates macrophage functions: migration, phagocytosis. • Tissue-type plasminogen activator via LRP1 modulates TLR function. • Plasminogen/plasmin regulate efferocytosis and macrophage M1/M2 polarization during inflammation resolution. • Plasmin inhibition can suppress the cytokine storm syndrome associated with macrophage activation syndrome.

We report a case of acute eosinophilic pneumonia (AEP) triggered by coronavirus disease 2019 (COVID‐19) infection. A 77‐year‐old man experienced left‐sided chest pain and shortness of breath. Reverse transcription‐polymerase chain reaction (RT‐PCR) for severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) revealed a positive result, and he was treated with favipiravir, ciclesonide, and lascufloxacin, but he showed poor improvement. On the other hand, computed tomography (CT) images were atypical for COVID‐19 infection, and the elevation of eosinophil was found in blood and the fluid obtained by bronchoscopy. So, we clinically diagnosed this case as AEP. Administration of prednisolone dramatically improved the patient's clinical condition and chest radiograph findings, which were consistent with the clinical course of AEP. This case suggests the importance of considering the complications of AEP when treating patients with COVID‐19 infection., We report a case of acute eosinophilic pneumonia (AEP) triggered by coronavirus disease 2019 (COVID‐19) infection. This case suggests the importance of considering the complications of AEP when treating patients with COVID‐19 infection.

Characterization of the target search dynamics of DNA-binding proteins along DNA has been hampered by the time resolution of a standard single-molecule fluorescence microscopy. Here, we achieved the time resolution of 0.5 ms in the fluorescence microscopy measurements by optimizing the fluorescence excitation based on critical angle illumination and by utilizing the time delay integration mode of the electron-multiplying charge coupled device. We characterized the target search dynamics of the tumor suppressor p53 along nonspecific DNA at physiological salt concentrations. We identified a short-lived encounter intermediate before the formation of the long-lived p53–DNA complex. Both the jumps and the one-dimensional diffusion of p53 along DNA were accelerated at higher salt concentrations, suggesting the rotation-uncoupled movement of p53 along DNA grooves and conformational changes in the p53/DNA complex. This method can be used to clarify the unresolved dynamics of DNA-binding proteins previously hidden by time averaging.

The self-assembly processes of two kinds of octahedron-shaped M6L4 cages consisting of cis-protected Pd(II) complexes and organic tritopic ligands were investigated. Whether kinetically trapped species larger than the cages are produced or the M6L4 cage is assembled without the formation of such kinetic traps is determined by a balance between the rates of oligomerization and intramolecular cyclization, which is affected by slight changes in the chemical structure of the tritopic ligand. A numerical analysis of the experimental data based on a reaction network model where 249 reactions between the possible 56 species were considered revealed the self-assembly pathways of one of the two M6L4 cages.

Self-assembly processes of three octahedron-shaped [Pd6L12]12+ cages were investigated by an NMR-based quantitative approach. As to the on-pathway of the Pd6L12 cage assembly, the final intramolecular ligand exchange in an incomplete cage, [Pd6L12Py*]12+ (Py*: 3-chloropyridine, which was used as a leaving ligand), is the rate-determining step in the self-assembly of all the three [Pd6L12]12+ cages. Contrary to the previous finding that the self-assembly of [PdmL2m]2m+ structures (m = 2, 3) and [Pd6L8]12+ capsules from rigid multitopic ligands efficiently takes place without the formation of kinetically trapped species under mild conditions, in the self-assembly of the [Pd6L12]12+ cages, even relatively rigid ditopic ligands co-produced 100 nm-sized kinetic traps through off-pathways, which would be because the energy landscape becomes more complicated by increasing the number of components in the final assembly. It was found that when Py* was used as a leaving ligand in CD3CN, the [Pd6L12]12+ cages were produced in high yield, preventing the formation of the kinetically trapped species, which indicates that the use of Py* as a leaving ligand in CD3CN is effective to obtain the thermodynamically most stable species.

The coordination self-assembly process of a Pd2L4 cage including rigid ditopic ligands, L, was studied numerically. A recently developed experimental approach (QASAP: quantitative analysis of self-assembly process) revealed that the rate-determining steps in the self-assembly of the Pd2L4 cage are intramolecular ligand exchanges at the late stages of the self-assembly. In this study, the self-assembly process before the rate-determining steps, which could not be investigated by experiment, was analyzed based on a minimal reaction network model. Only eight variable parameters of rate constants for ligand exchange reactions are sufficient enough to reproduce the time evolution of substrates and the products during the self-assembly of the cage. With these parameters, the major self-assembly pathway was determined. It was also found that a non-negligible amount of an incomplete cage (IC), Pd2L3X2 (X indicates the leaving ligand), which was not suggested by QASAP, should be transiently produced. Numerical tests also suggest that the small rate constant value of the intramolecular ligand exchanges due to a restricted geometry causes the final stage to seemingly become the rate-determining step.

Angiogenic factors play a key role in multiple myeloma (MM) growth, relapse, and drug resistance. Here we show that malignant plasma cells (cell lines and patient-derived MM cells) express angiocrine factor EGF like-7 (EGFL7) mRNA and protein. MM cells both produced EGFL7 and expressed the functional EGFL7 receptor integrin β 3 (ITGB3), resulting in ITGB3 phosphorylation and focal adhesion kinase activation. Overexpression of ITGB3 or EGFL7 enhanced MM cell adhesion and proliferation. Intriguingly, ITGB3 overexpression upregulated the transcription factor Krüppel-like factor 2 (KLF2), which further enhanced EGFL7 transcription in MM cells, thereby establishing an EGFL7-ITGB3-KLF2-EGFL7 amplification loop that supports MM cell survival and proliferation. EGFL7 expression was found in certain plasma cells of patients with refractory MM and of patients at primary diagnosis. NOD.CB17-Prkdc/J mice transplanted with MM cells showed elevated human plasma EGFL7 levels. EGFL7 knockdown in patient-derived MM cells and treatment with neutralizing antibodies against EGFL7 inhibited MM cell growth in vitro and in vivo. We demonstrate that the standard-of-care MM drug bortezomib upregulates EGFL7, ITGB3, and KLF2 expression in MM cells. Inhibition of EGFL7 signaling in synergy with BTZ may provide a novel strategy for inhibiting MM cell proliferation.

The self-assembly process of a Pd4L8 double-walled square (DWS) was investigated. As was seen in Pd2L4 cages, Pd(II)-linked coordination self-assembly processes are generally affected by the rigidity of multitopic ligands. However, the self-assembly of a Pd4L8 DWS from rigid ditopic ligands took place with the formation of two kinds of metastable species [submicrometer-sized species and a Pd3L6 double-walled triangle (DWT)]. This result suggests that the self-assembly process of the DWS is largely affected by the geometry of the final product and not by that of the ditopic ligand.

The kinetics on a basic ligand substitution reaction on dinuclear platinum complexes [Pt(PEt3 )2 PhPt(PEt3 )2 ]2+ and [Pt(PEt3 )2 PhCOPhPt(PEt3 )2 ]2+ , with the ligands pyridine and 3-chloropyridine, is studied. This is a fundamental step in a self-assembly, and the time evolution has been observed with a new experimental technique, QASAP (quantitative analysis of self-assembly process), which is recently developed by Hiraoka's group. As a result of numerical calculations based on master equation, we succeed in specifying the reaction rate constants with a simple reaction model. In addition, the time evolutions of all the intermediate components produced and consumed in chemical reaction are revealed, including those unobserved in the experiments. The convergence behavior of the existence ratios of specific chemical species calculated with the stochastic algorithm method is compared with those obtained from deterministic formalism based on rate equations, revealing a clear dependence on the number of constituent molecules. © 2018 Wiley Periodicals, Inc.

Intersegmental transfer (IST) is an important strategy in the target search used by sequence-specific DNA-binding proteins (DBPs), enabling DBPs to search for targets between multiple DNA strands without dissociation. We examined the IST of the tumor suppressor p53 using ensemble stopped-flow and single-molecule fluorescence measurements. The ensemble measurements demonstrated that p53 exhibits very fast IST, whose rate constant was ∼108 M−1 s−1. To determine the domains of p53 responsible for IST, two mutants with deletions of one of its two DNA binding domains were generated. The mutant lacking the disordered C-terminal (CT) domain (the CoreTet mutant) abolished IST, whereas the mutant lacking the structured core domain (the TetCT mutant) maintained IST, clearly demonstrating the importance of the CT domain. Single-molecule fluorescence measurements further demonstrated the transfer of p53 between two tethered DNA strands. The pseudo-wild-type p53 and the TetCT mutant showed significant transfer efficiencies, whereas the transfer efficiency for the CoreTet mutant was zero. These results suggest that ultrafast IST might be promoted by four copies of the CT domain, by binding to two DNA strands simultaneously. Such ultrafast IST might be important to avoid nearby-bound DBPs during the target search process of p53 in nucleus.

We propose a hypothesis that explains two apparently contradicting observations for the heterogeneity of the unfolded proteins. First, the line confocal method of the single-molecule Förster resonance energy transfer (sm-FRET) spectroscopy revealed that the unfolded proteins possess broad peaks in the FRET efficiency plot, implying the significant heterogeneity that lasts longer than milliseconds. Second, the fluorescence correlation method demonstrated that the unfolded proteins fluctuate in the time scale shorter than 100 ns. To formulate the hypothesis, we first summarize the recent consensus for the structure and dynamics of the unfolded proteins. We next discuss the conventional method of the sm-FRET spectroscopy and its limitations for the analysis of the unfolded proteins, followed by the advantages of the line confocal method that revealed the heterogeneity. Finally, we propose that the structural heterogeneity formed by the local clustering of hydrophobic residues modulates the distribution of the long-range distance between the labeled chromophores, resulting in the broadening of the peak in the FRET efficiency plot. A clustering of hydrophobic residues around the chromophore might further contribute to the broadening. The proposed clusters are important for the understanding of protein folding mechanism.

PURPOSE Fluorescence imaging is an important surgical adjunct in malignant glioma surgery. 5-aminolevulinic acid (5-ALA) has been proven effective for radical tumor resection and extended progression-free survival in a phase III randomized trial and therefore integrated into surgery for malignant glioma. Importantly, however, some limitations still exist in its use, which include false positivity and false negativity as well as inability of re-administration. In this study, we aimed to develop a novel, spray-type fluorescent probe using hydroxymethyl rhodamine green (HMRG) as a fluorescent scaffold. METHODS We have previously established a fluorescent probe library comprised of more than 320 kinds of HMRG probes. They have HMRG as a fluorescent scaffold with various types of dipeptides attached to it. Primary probe screening was performed using the homogenized tumor samples from patients with glioblastoma operated at our institution. Secondary screening followed using the selected probes and fresh tumor samples obtained from patients with glioblastoma operated from 2016 until 2018. Diced electrophoresis gel (DEG) assay, two-dimensional gel electrophoresis followed by a multi-well plate-based fluorometric assay, was performed to identify responsible enzymes for the selected probe. Further experiments with inhibitors, real-time PCR, immunohistochemistry, and western blotting were performed for confirmation. RESULTS Proline-arginine-HMRG (PR-HMRG) was selected as a candidate probe based upon the above two-step screenings. It achieved 79.4% accuracy in receiver operating characteristic curve analysis. Calpain-1 was found to be responsible to cleave PR-HMRG probe by DEG-proteome analysis. Calpain-1 protein was expressed at significantly higher level in tumors that were fluoresced by PR-HMRG than in those that were not. CONCLUSIONS Our innovative screening method was able to find PR-HMRG as a novel fluorescent probe effective for rapid detection of glioblastoma. A preclinical study is planned to assess the efficacy and safety of the selected probe.

DNA methylation is an epigenetic gene regulatory mechanism that plays an essential role in gene expression, transposon silencing, genome imprinting and plant development. We investigated the influence of DNA methylation on gene expression in Brassica rapa L., to understand whether epigenetic differences exist between inbred lines. Genome-wide DNA methylation was analysed by methylated DNA immunoprecipitation sequencing (MeDIP-seq) of 14-day-old first and second leaves from two inbred lines of Chinese cabbage, one susceptible and one resistant to fusarium yellows caused by Fusarium oxysporum f. sp. conglutinans. MACS (model-based analysis for ChIP-seq) identified DNA methylation peaks in genic regions including 2 kb upstream, exon, intron and 2 kb downstream. More than 65% of genes showed similar patterns of DNA methylation in the genic regions in the two inbred lines. DNA methylation states of the two inbred lines were compared with their transcriptome. Genes having DNA methylation in the intron and in the 200 bp upstream and downstream regions were associated with a lower expression level in both lines. A small number of genes showed a negative correlation between differences in DNA methylation levels and differences in transcriptional levels in the two inbred lines, suggesting that DNA methylation in these genes results in transcriptional suppression.

The tumor suppressor p53 is a multidomain transcription factor that can quickly bind to its target DNA by sliding along the DNA strand. We hypothesized that the intrinsically disordered and positively charged linker of p53 regulates its search dynamics first by directly interacting with DNA and second by modulating hopping of the core domain. To test the two hypotheses, we prepared five variants of p53 in which the length and charge of the linker were modulated. The affinity for and sliding along nonspecific DNA of p53 were altered by the charge of the linker, but not by the linker length. In particular, charge neutralization significantly reduced the affinity, suggesting that the linker directly contacts the DNA. Charge neutralization eliminated the slow mode of sliding, in which the core domain was assumed to contact nonspecific DNA. In contrast, the affinity of p53 for the target DNA was not affected by linker mutations. These results demonstrate that the linker participates in a target search of p53 by contacting nonspecific DNA and recruiting the core domain to contact DNA.

Tumor suppressor p53 slides along DNA and finds its target sequence in drastically different and changing cellular conditions. To elucidate how p53 maintains efficient target search at different concentrations of divalent cations such as Ca2+ and Mg2+, we prepared two mutants of p53, each possessing one of its two DNA-binding domains, the CoreTet mutant having the structured core domain plus the tetramerization (Tet) domain, and the TetCT mutant having Tet plus the disordered C-terminal domain. We investigated their equilibrium and kinetic dissociation from DNA and search dynamics along DNA at various [Mg2+]. Although binding of CoreTet to DNA becomes markedly weaker at higher [Mg2+], binding of TetCT depends slightly on [Mg2+]. Single-molecule fluorescence measurements revealed that the one-dimensional diffusion of CoreTet along DNA consists of fast and slow search modes, the ratio of which depends strongly on [Mg2+]. In contrast, diffusion of TetCT consisted of only the fast mode. The disordered C-terminal domain can associate with DNA irrespective of [Mg2+], and can maintain an equilibrium balance of the two search modes and the p53 search distance. These results suggest that p53 modulates the quaternary structure of the complex between p53 and DNA under different [Mg2+] and that it maintains the target search along DNA.

Elucidating the protein folding mechanism is crucial to understand how proteins acquire their unique structures to realize various biological functions. With this aim, the folding/unfolding of small globular proteins has been extensively studied. Interestingly, recent studies have revealed that even such small proteins represent considerably complex processes. In this study, we examined the folding/unfolding process of a small α-helical protein, the B domain of protein A (BdpA), at equilibrium using two-dimensional fluorescence lifetime correlation spectroscopy with 10 μs time resolution. The results showed that although the BdpA is a two-state folder, both the native and unfolded states are highly heterogeneous and the conformational conversion within each ensemble occurs within 10 μs. Furthermore, it was shown that the average structures of both ensembles gradually change and become more elongated as the denaturant concentration increases. The analysis on two mutants suggested that fraying of the N-terminal helix is the origin of the inhomogeneity of the native state. Because the direct observation of the ensemble nature of the native state at the single-molecule level has not been reported, the data obtained in this study give new insights into complex conformational properties of small proteins.

ATP-binding cassette (ABC) transporters are ubiquitously present in prokaryotic and eukaryotic cells. Binding of ATP to the nucleotide-binding domains (NBDs) elicits major conformational changes of the transporters resulting in the transport of the substrate across the membrane. The availability of a crystal structure of the NBDs enabled us to elucidate the local structure and small-scale dynamics in the NBDs. Here, we labeled the ABC transporter MsbA, a homodimeric flippase from Escherichia coli, with a fluorescent probe, Alexa532, within the NBDs. ATP application elicited collisional quenching, whereas no quenching was observed after the addition of ATP analogs or ATP hydrolysis inhibitors. The Alexa532-conjugated MsbA variants exhibited transition metal ion Förster resonance energy transfer (tmFRET) after the addition of Ni2+, and ATP decreased this Ni2+-mediated FRET of the NBDs. Structure modeling developed from crystallographic data and examination of tmFRET measurements of MsbA variants in the absence of ATP revealed the presence of metal ion-associated pockets (MiAPs) in the NBDs. Three histidines were predicted to participate in chelating Ni2+ in the two possible MiAPs. Performing histidine-substitution experiments with the NBDs showed that the dissociation constant for Ni2+ of MiAP2 was smaller than that of MiAP1. The structural allocation of the MiAPs was further supported by showing that the addition of Cu2+ resulted in higher quenching than Ni2+. Taken together, the present study showed that the NBDs contain two native binding sites for metal ions and ATP addition affects the Ni2+-binding activity of the MiAPs.

Sequence-specific DNA-binding proteins can maintain and regulate cellular functions by accurately and quickly binding to target sequences among large amounts of nontarget DNA. The facilitated diffusion mechanism of DNA-binding proteins—a combination of three-dimensional (3D) diffusion and one-dimensional (1D) sliding along DNA—has been proposed to explain the target binding accuracy and rapidity and has been partially confirmed experimentally. Nonetheless, quantitative elucidation of the mechanism has remained difficult. Furthermore, many additional steps in facilitated diffusion have been proposed. In this review, we introduce the theoretical and experimental studies and the current understanding of facilitated diffusion of DNA-binding proteins. We focused on tumor suppressor p53 as a key protein subject to facilitated diffusion; p53 regulates various cellular processes such as cell cycle arrest, DNA repair, and apoptosis upon binding to a target sequence of DNA after activation by external stress to the cell. We describe the research on the 3D diffusion and 1D sliding of p53 mainly via single-molecule fluorescence microscopy. In addition to the demonstration of the 1D sliding of p53, recent experiments revealed multiple modes of 1D sliding, regulation of the target recognition, and the constant search distance despite changes in the concentrations of divalent cations. Furthermore, rotation-coupled 1D sliding along DNA is suggested. A comparison of parameters of the facilitated diffusion of p53 and those of other DNA-binding proteins characterized so far suggests that the ratio of 3D diffusion and 1D sliding is close to the theoretical optimum of 1:1 for several proteins including p53.

The template effect is a key feature to control the arrangement of building blocks in assemblies, but its kinetic nature remains elusive compared to the thermodynamic aspects, with the exception of very simple reactions. Here we report a kinetic template effect in a self-assembled cage composed of flexible ditopic ligands and Pd(II) ions. Without template anion, a micrometer-sized sheet is kinetically trapped (off-pathway), which is converted into the thermodynamically most stable cage by the template anion. When the template anion is present from the start, the cage is selectively produced by the preferential cyclization of a dinuclear intermediate (on-pathway). Quantitative and numerical analyses of the self-assembly of the cage on the on-pathway revealed that the accelerating effect of the template is stronger for the early stage reactions of the self-assembly than for the final cage formation step itself, indicating the kinetic template effect. Thermodynamic template effects are widely used in supramolecular assembly, but kinetic template effects are less well understood. Here the contributions of thermodynamic and kinetic template effects in the self-assembly of palladium metallocages are disentangled using QASAP methodology.

BACKGROUND Chordoma and chondrosarcoma account for the majority of skull base tumors affecting the petroclival region. Chondrosarcoma has better prognosis than chordoma; heavy particle therapy is often indicated for residual/recurrent chordoma. Preoperative, precise diagnosis of the tumor would be desirable, as it can potentially impact on the choice of a surgical approach and the aggressiveness of surgery. METHODS We conducted a radiomics study to create a machine learning model distinguishing chondrosarcoma from chordoma. We collected DICOM T2-weighted images and T1-weighted images with gadolinium (GdT1) enhancement in the consective patients of chordoma or chondrosarcoma who underwent surgery at The University of Tokyo Hospital from September of 2012 to January of 2019. We selected patients with uniform MRI images. VOI (volume of interest) was set using Monaco (https://www.elekta.com/software-solutions/treatment-management/external-beam-planning/monaco.html). Not only sematic features but also agnostic features were calculated. The original images and 8 wavelet transformed images were calculated for texture agnostic features such as Gray-Level Co-occurrence Matrix (GLCM). Features were selected by recursive feature elimination (RFE). The final model evaluation was performed by average area under the curve (AUC). RESULTS The study population included 17 chordomas and 22 chondrosarcomas in a total of 39 patients. 476 features were obtained per image sequence. The number of features per case was 476 × 2 = 952 The most accurate machine learning model was created using the extracted three features from only T2. The best AUC was 0.77 ± 0.11 in logistic regression (dataset was divided randomly into halves, average value of AUC calculated six times). CONCLUSIONS This novel machine learning model can differentiate chordoma and chondrosarcoma reasonably well. A validation study with a larger number of patients is warranted.

PURPOSE: ALA is commonly used as an intraoperative tool in malignant glioma surgery, which has been proven effective for radical tumor resection and extended progression-free survival. However, there are some limitations in its use, such as false positivity, false negativity, and inability of re-administration. We aim to develop a novel fluorescent labeling system which can be repeatedly administered by spray during surgery, using hydroxymethyl rhodamine green (HMRG) as fluorescent scaffold originally designed at our university for cancer detection. [Methods]Primary probe screening was performed using the homogenized glioblastoma (GBM) samples with the fluorescent probe library comprised of more than 320 kinds of HMRG fluorescent scaffold combined with various types of dipeptides. Second probe screening was performed using fresh GBM specimens and the selected probes in primary screening. To identify the responsible enzymes, diced electrophoresis gel (DEG) assay was performed. This method utilizes the combination of two dimensional electrophoresis (isoelectric point and molecular weight) and a multiwell-plate-based fluorometric assay to find protein spots with the specified activities. [Results] The prominent probes were selected based upon the above two-step screenings. We identified two enzymes by proteome analysis and experiments using inhibitors, which was further confirmed with real-time PCR and western blotting. [Discussion] This screening methodology is innovative in that it is based on selecting probes from the probe library that respond to clinical samples rather than creating probes from the responsible enzymes. Practical fluorescent probes can be established even for low-grade gliomas, which would be a breakthrough for rapid intraoperative diagnosis in glioma surgery. [Conclusion] HMRG-based aminopeptidase fluorescent probes may be effective for GBM detection.

Early in vivo studies demonstrated the involvement of a tumor-suppressing transcription factor, p53, into cellular droplets such as Cajal and promyelocytic leukemia protein bodies, suggesting that the liquid-liquid phase separation (LLPS) might be involved in the cellular functions of p53. To examine this possibility, we conducted extensive investigations on the droplet formation of p53 in vitro. First, p53 itself was found to form liquid-like droplets at neutral and slightly acidic pH and at low salt concentrations. Truncated p53 mutants modulated droplet formation, suggesting the importance of multivalent electrostatic interactions among the N-terminal and C-terminal domains. Second, FRET efficiency measurements for the dimer mutants of p53 revealed that distances between the core domains and between the C-terminal domains were modulated in an opposite manner within the droplets. Third, the molecular crowding agents were found to promote droplet formation, whereas ssDNA, dsDNA, and ATP, to suppress it. Finally, the p53 mutant mimicking posttranslational phosphorylation did not form the droplets. We conclude that p53 itself has a potential to form droplets that can be controlled by cellular molecules and by posttranslational modifications, suggesting that LLPS might be involved in p53 function.

The external application of acetic acid has recently been reported to enhance survival of drought in plants such as Arabidopsis, rapeseed, maize, rice, and wheat, but the effects of acetic acid application on increased drought tolerance in woody plants such as a tropical crop “cassava” remain elusive. A molecular understanding of acetic acid-induced drought avoidance in cassava will contribute to the development of technology that can be used to enhance drought tolerance, without resorting to transgenic technology or advancements in cassava cultivation. In the present study, morphological, physiological, and molecular responses to drought were analyzed in cassava after treatment with acetic acid. Results indicated that the acetic acid-treated cassava plants had a higher level of drought avoidance than water-treated, control plants. Specifically, higher leaf relative water content, and chlorophyll and carotenoid levels were observed as soils dried out during the drought treatment. Leaf temperatures in acetic acid-treated cassava plants were higher relative to leaves on plants pretreated with water and an increase of ABA content was observed in leaves of acetic acid-treated plants, suggesting that stomatal conductance and the transpiration rate in leaves of acetic acid-treated plants decreased to maintain relative water contents and to avoid drought. Transcriptome analysis revealed that acetic acid treatment increased the expression of ABA signaling-related genes, such as OPEN STOMATA 1 (OST1) and protein phosphatase 2C; as well as the drought response and tolerance-related genes, such as the outer membrane tryptophan-rich sensory protein (TSPO), and the heat shock proteins. Collectively, the external application of acetic acid enhances drought avoidance in cassava through the upregulation of ABA signaling pathway genes and several stress responses- and tolerance-related genes. These data support the idea that adjustments of the acetic acid application to plants is useful to enhance drought tolerance, to minimize the growth inhibition in the agricultural field.

Touch shape discrimination is not only closely related to tactile mechanoreceptors but also higher cognitive function. However, previous shape discrimination methods are difficult to complete in a short time, and the devices are complicated to operate and not user-friendly for nonprofessionals. Here, we propose a new method, the evaluation quantity of which is the angle discrimination threshold. In addition, to make this method easy to use for nonprofessionals, we designed a haptic angle sorting system, including the device and software. To evaluate this method, the angle sorting and two-angle discrimination experiments were compared, and it was found that participants spent significantly less time in the former experiment than in the latter. At the same time, there is a strong correlation between the performance of angle sorting and two-angle discrimination, which shows that the angle threshold obtained by the new method can also be used to evaluate the ability of touch discrimination. Moreover, the angle sorting results of different age groups also further demonstrate the feasibility of the method. The efficiency of this new method and the effectiveness of the system also provide a convenient means for evaluating haptic shape discrimination, which may have potential clinical application value in the early diagnosis of peripheral neuropathy and even in the evaluation of cognitive function.

The proton yields from Si(111)7 × 7 and Si(100)2 × 1 reconstructed surfaces irradiated with slow ( v 0.25 v Bohr ) highly charged Xe ions are obtained. H 2 O molecules adsorbed over time on the surfaces at room temperature, under ultra-high vacuum can be detected as an increase of the proton yield. For the Si(100)2 × 1 surface, the proton yield with HCI-irradiation time is discussed based on temporal variation of the H 2 O coverage. The proton desorption efficiency with Xe 50 + is more than about ten times and twice as compared with Xe 29 + and Xe 44 + , respectively. For the Si(111)7 × 7 and Si(100)2 × 1 surfaces, the proton yield in each time increases with the charge states q to the power of 6 and 4, respectively, and is changed with time with the power laws held on.