Validation of a New Methodology to Create Oral Drugs beyond the Rule of 5 for Intracellular Tough Targets.

Establishing a technological platform for creating clinical compounds inhibiting intracellular protein-protein interactions (PPIs) can open the door to many valuable drugs. Although small molecules and antibodies are mainstream modalities, they are not suitable for a target protein that lacks a deep cavity for a small molecule to bind or a protein found in intracellular space out of an antibody's reach. One possible approach to access these targets is to utilize so-called middle-size cyclic peptides (defined here as those with a molecular weight of 1000-2000 g/mol). In this study, we validated a new methodology to create oral drugs beyond the rule of 5 for intracellular tough targets by elucidating structural features and physicochemical properties for drug-like cyclic peptides and developing library technologies to afford highly N-alkylated cyclic peptide hits. We discovered a KRAS inhibitory clinical compound (LUNA18) as the first example of our platform technology.

Tracheary elements from calli of Japanese horse chestnut (Aesculus turbinata) form perforation-like structures.

MAIN CONCLUSION: Calli derived from young leaves of Aesculus turbinata contained tracheary elements with large pores that resembled perforations of vessel elements. The differentiation of tracheary elements in vitro provides a useful system for detailed analysis of xylem cell differentiation. To examine the mechanism of formation of cell wall structures, new differentiation systems are required that allows us to induce highly organized structures, such as perforations. In this study, we developed such a system in which we were able to induce formation of tracheary elements with perforations, using calli of a hardwood, Aesculus turbinata. Young leaves of A. turbinata were placed on modified MS medium that contained 5 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5 µM benzyladenine (BA). Tracheary elements were induced in calli derived from young leaves of A. turbinata. Some tracheary elements formed broad areas of secondary wall with typical features of secondary xylem. Other tracheary elements formed spiral thickenings, which are typical features of vessel elements in secondary xylem of A. turbinata. Approximately 10% of tracheary elements formed large pores that resembled perforations of vessel elements and various types of the perforation plate were observed. Addition of NAA and brassinolide to the induction medium enhanced the differentiation of tracheary elements in calli of A. turbinata. Newly induced tracheary elements also formed typical features of secondary xylem such as perforations of the vessel elements. Our model system might be useful in efforts to understand the mechanisms of formation of highly organized structures in tracheary elements in secondary xylem.

Winter-spring temperature pattern is closely related to the onset of cambial reactivation in stems of the evergreen conifer Chamaecyparis pisifera.

Temperature is an important factor for the cambial growth in temperate trees. We investigated the way daily temperatures patterns (maximum, average and minimum) from late winter to early spring affected the timing of cambial reactivation and xylem differentiation in stems of the conifer Chamaecyparis pisifera. When the daily temperatures started to increase earlier from late winter to early spring, cambial reactivation occurred earlier. Cambium became active when it achieves the desired accumulated temperature above the threshold (cambial reactivation index; CRI) of 13 °C in 11 days in 2013 whereas 18 days in 2014. This difference in duration required for achieving accumulated temperature can be explained with the variations in the daily temperature patterns in 2013 and 2014. Our formula for calculation of CRI predicted the cambial reactivation in 2015. A hypothetical increase of 1-4 °C to the actual daily maximum temperatures of 2013 and 2014 shifted the timing of cambial reactivation and had different effects on cambial reactivation in the two consecutive years because of variations in the actual daily temperatures patterns. Thus, the specific annual pattern of accumulation of temperature from late winter to early spring is a critical factor in determining the timing of cambial reactivation in trees.

Artificially lignified cell wall catalyzed by peroxidase selectively localized on a network of microfibrils from cultured cells.

MAIN CONCLUSION: An artificial lignified cell wall was synthesized in three steps: (1) isolation of microfibrillar network; (2) localization of peroxidase through immunoreaction; and (3) polymerization of DHP to lignify the cell wall. Artificial woody cell wall synthesis was performed following the three steps along with the actual formation in nature using cellulose microfibrils extracted from callus derived from Cryptomeria japonica. First, we constructed a polysaccharide network on a transmission electron microscopy (TEM) grid. The preparation method was optimized by chemical treatment, followed by mechanical fibrillation to create a microfibrillated network. Morphology was examined by TEM, and chemical characterization was by Fourier transform infrared (FTIR) spectroscopy. Second, we optimized the process to place peroxidase on the microfibrils via an immunoreaction technique. Using a xyloglucan antibody, we could ensure that gold particles attached to the secondary antibodies were widely and uniformly localized along with the microfibril network. Third, we applied the peroxidase attached to secondary antibodies and started to polymerize the lignin on the grid by simultaneously adding coniferyl alcohol and hydrogen peroxide. After 30 min of artificial lignification, TEM observation showed that lignin-like substances were deposited on the polysaccharide network. In addition, FTIR spectra revealed that the bands specific for lignin had increased, demonstrating the successful artificial formation of woody cell walls. This approach may be useful for studying woody cell wall formation and for producing made-to-order biomaterials.

Continued growth of locally aggressive fibrous dysplasia of 22 years duration after reaching adulthood: a case report.

Fibrous dysplasia generally stops growing when patients reach adulthood. Locally aggressive fibrous dysplasia is an extremely rare subtype of fibrous dysplasia that is characterized by progressive enlargement after bone maturation, cortical bone destruction and soft tissue invasion but without malignant transformation. At 50 years of age, a tumor was found in the rib of a patient. The tumor gradually enlarged over time and imaging findings suggested a malignant tumor. The case was further complicated by restrictive lung disorder. Biopsies from multiple sites showed no malignant findings, and marginal resection with partial curettage was performed. The final diagnosis was locally aggressive fibrous dysplasia, and the restrictive lung disorder improved postoperatively. The natural history of the disease is also unknown. This is the first report in the literature to describe a case in which a lesion exhibited long-term growth over a period of 22 years after reaching adulthood.

Changes in cambial activity are related to precipitation patterns in four tropical hardwood species grown in Indonesia.

PREMISE: Cambial activity in some tropical trees varies intra-annually, with the formation of xylem rings. Identification of the climatic factors that regulate cambial activity is important for understanding the growth of such species. We analyzed the relationship between climatic factors and cambial activity in four tropical hardwoods, Acacia mangium, Tectona grandis, Eucalyptus urophylla, and Neolamarckia cadamba in Yogyakarta, Java Island, Indonesia, which has a rainy season (November-June) and a dry season (July-October). METHODS: Small blocks containing phloem, cambium, and xylem were collected from main stems in January 2014, October 2015 and October 2016, and examined with light microscopy for cambial cell division, fusiform cambial cells, and expanding xylem cells as evidence of cambial activity. RESULTS: During the rainy season, when precipitation was high, cambium was active. By contrast, during the dry season in 2015, when there was no precipitation, cambium was dormant. However, in October 2016, during the so-called dry season, cambium was active, cell division was conspicuous, and a new xylem ring formation was initiated. The difference in cambial activity appeared to be related to an unusual pattern of precipitation during the typically dry months, from July to October, in 2016. CONCLUSIONS: Our results indicate that low or absent precipitation for 3 to 4 months induces cessation of cambial activity and temporal periodicity of wood formation in the four species studied. By contrast, in the event of continuing precipitation, cambial activity in the same trees may continue throughout the year. The frequency pattern of precipitation appears to be an important determinant of wood formation in tropical trees.

Stem gravitropism and tension wood formation in Acacia mangium seedlings inclined at various angles.

Background and Aims: In response to a gravitational stimulus, angiosperm trees generally form tension wood on the upper sides of leaning stems in order to reorientate the stems in the vertical direction. It is unclear whether the angle of inclination from the vertical affects tension wood formation. This study was designed to investigate negative gravitropism, tension wood formation and growth eccentricity in Acacia mangium seedlings inclined at different angles. Methods: Uniform seedlings of A. mangium were artificially inclined at 30°, 45°, 60° and 90° from the vertical and harvested, with non-inclined controls, 3 months later. We analysed the effects of the angle of inclination on the stem recovery angle, the anatomical features of tension wood and radial growth. Key Results: Smaller inclination angles were associated with earlier stem recovery while stems subjected to greater inclination returned to the vertical direction after a longer delay. However, in terms of the speed of negative gravitopism towards the vertical, stems subjected to greater inclination moved more rapidly toward the vertical. There was no significant difference in terms of growth eccentricity among seedlings inclined at different angles. The 30°-inclined seedlings formed the narrowest region of tension wood but there were no significant differences among seedlings inclined at 45°, 60° and 90°. The 90°-inclined seedlings formed thicker gelatinous layers than those in 30°-, 45°- and 60°-inclined seedlings. Conclusion: Our results suggest that the angle of inclination of the stem influences negative gravitropism, the width of the tension wood region and the thickness of gelatinous layers. Larger amounts of gelatinous fibres and thicker gelatinous layers might generate the higher tensile stress required for the higher speed of stem-recovery movement in A. mangium seedlings.

Cavitation of intercellular spaces is critical to establishment of hydraulic properties of compression wood of Chamaecyparis obtusa seedlings.

BACKGROUND AND AIMS: When the orientation of the stems of conifers departs from the vertical as a result of environmental influences, conifers form compression wood that results in restoration of verticality. It is well known that intercellular spaces are formed between tracheids in compression wood, but the function of these spaces remains to be clarified. In the present study, we evaluated the impact of these spaces in artificially induced compression wood in Chamaecyparis obtusa seedlings. METHODS: We monitored the presence or absence of liquid in the intercellular spaces of differentiating xylem by cryo-scanning electron microscopy. In addition, we analysed the relationship between intercellular spaces and the hydraulic properties of the compression wood. KEY RESULTS: Initially, we detected small intercellular spaces with liquid in regions in which the profiles of tracheids were not rounded in transverse surfaces, indicating that the intercellular spaces had originally contained no gases. In the regions where tracheids had formed secondary walls, we found that some intercellular spaces had lost their liquid. Cavitation of intercellular spaces would affect hydraulic conductivity as a consequence of the induction of cavitation in neighbouring tracheids. CONCLUSIONS: Our observations suggest that cavitation of intercellular spaces is the critical event that affects not only the functions of intercellular spaces but also the hydraulic properties of compression wood.

Localized cooling of stems induces latewood formation and cambial dormancy during seasons of active cambium in conifers.

BACKGROUND AND AIMS: In temperate regions, trees undergo annual cycles of cambial growth, with periods of cambial activity and dormancy. Environmental factors might regulate the cambial growth, as well as the development of cambial derivatives. We investigated the effects of low temperature by localized cooling on cambial activity and latewood formation in two conifers, Chamaecyparis obtusa and Cryptomeria japonica. METHODS: A plastic rubber tube that contained cooled water was wrapped around a 30-cm-wide portion of the main stem of Chamaecyparis obtusa and Cryptomeria japonica trees during seasons of active cambium. Small blocks were collected from both cooled and non-cooled control portions of the stems for sequential observations of cambial activity and for anatomical measurements of cell morphology by light microscopy and image analysis. KEY RESULTS: The effect of localized cooling was first observed on differentiating tracheids. Tracheids narrow in diameter and with significantly decreased cambial activity were evident 5 weeks after the start of cooling in these stems. Eight weeks after the start of cooling, tracheids with clearly diminished diameters and thickened cell walls were observed in these stems. Thus, localized low temperature induced narrow diameters and obvious thickening of secondary cell walls of tracheids, which were identified as latewood tracheids. Two months after the cessation of cooling, a false annual ring was observed and cambium became active again and produced new tracheids. In Cryptomeria japonica, cambial activity ceased earlier in locally cooled portions of stems than in non-cooled stems, indicating that the cambium had entered dormancy sooner in the cooled stems. CONCLUSIONS: Artificial cooling of stems induced latewood formation and cessation of cambial activity, indicating that cambium and its derivatives can respond directly to changes in temperature. A decrease in the temperature of the stem is a critical factor in the control of cambial activity and xylem differentiation in trees.

The effects of localized heating and disbudding on cambial reactivation and formation of earlywood vessels in seedlings of the deciduous ring-porous hardwood, Quercus serrata.

BACKGROUND AND AIMS: The networks of vessel elements play a vital role in the transport of water from roots to leaves, and the continuous formation of earlywood vessels is crucial for the growth of ring-porous hardwoods. The differentiation of earlywood vessels is controlled by external and internal factors. The present study was designed to identify the limiting factors in the induction of cambial reactivation and the differentiation of earlywood vessels, using localized heating and disbudding of dormant stems of seedlings of a deciduous ring-porous hardwood, Quercus serrata. METHODS: Localized heating was achieved by wrapping an electric heating ribbon around stems. Disbudding involved removal of all buds. Three treatments were initiated on 1 February 2012, namely heating, disbudding and a combination of heating and disbudding, with untreated dormant stems as controls. Cambial reactivation and differentiation of vessel elements were monitored by light and polarized-light microscopy, and the growth of buds was followed. KEY RESULTS: Cambial reactivation and differentiation of vessel elements occurred sooner in heated seedlings than in non-heated seedlings before bud break. The combination of heating and disbudding of seedlings also resulted in earlier cambial reactivation and differentiation of first vessel elements than in non-heated seedlings. A few narrow vessel elements were formed during heating after disbudding, while many large earlywood vessel elements were formed in heated seedlings with buds. CONCLUSIONS: The results suggested that, in seedlings of the deciduous ring-porous hardwood Quercus serrata, elevated temperature was a direct trigger for cambial reactivation and differentiation of first vessel elements. Bud growth was not essential for cambial reactivation and differentiation of first vessel elements, but might be important for the continuous formation of wide vessel elements.

Gibberellin mediates the development of gelatinous fibres in the tension wood of inclined Acacia mangium seedlings.

BACKGROUND AND AIMS: Gibberellin stimulates negative gravitropism and the formation of tension wood in tilted Acacia mangium seedlings, while inhibitors of gibberellin synthesis strongly inhibit the return to vertical growth and suppress the formation of tension wood. To characterize the role of gibberellin in tension wood formation and gravitropism, this study investigated the role of gibberellin in the development of gelatinous fibres and in the changes in anatomical characteristics of woody elements in Acacia mangium seedlings exposed to a gravitational stimulus. METHODS: Gibberellin, paclobutrazol and uniconazole-P were applied to the soil in which seedlings were growing, using distilled water as the control. Three days after the start of treatment, seedlings were inclined at 45 ° to the vertical and samples were harvested 2 months later. The effects of the treatments on wood fibres, vessel elements and ray parenchyma cells were analysed in tension wood in the upper part of inclined stems and in the opposite wood on the lower side of inclined stems. KEY RESULTS: Application of paclobutrazol or uniconazole-P inhibited the increase in the thickness of gelatinous layers and prevented the elongation of gelatinous fibres in the tension wood of inclined stems. By contrast, gibberellin stimulated the elongation of these fibres. Application of gibberellin and inhibitors of gibberellin biosynthesis had only minor effects on the anatomical characteristics of vessel and ray parenchyma cells. CONCLUSIONS: The results suggest that gibberellin is important for the development of gelatinous fibres in the tension wood of A. mangium seedlings and therefore in gravitropism.

In vitro induction of secondary xylem-like tracheary elements in calli of hybrid poplar (Populus sieboldii × P. grandidentata).

The formation of tracheary elements was induced in calli derived from petioles of hybrid poplar (Populus sieboldii × P. grandidentata) after 10 days of culture on medium that lacked auxin but contained 1 µM brassinolide. Some differentiated cells formed broad regions of cell walls and bordered pits, which are typical features of tracheary elements of secondary xylem. Other differentiated cells resembled tracheary elements of primary xylem, with spiral or reticulate thickening of cell walls. The tracheary elements that developed in calli were formed within cell clusters. This induction system provides a new model for studies of the mechanism of differentiation of secondary xylem cells in vitro.

Regulation of cambial activity in relation to environmental conditions: understanding the role of temperature in wood formation of trees.

The timing of cambial reactivation plays an important role in determination of the amount and quality of wood and the environmental adaptivity of trees. Environmental factors, such as temperature, influence the growth and development of trees. Temperatures from late winter to early spring affect the physiological processes that are involved in the initiation of cambial cell division and xylem differentiation in trees. Cumulative elevated temperatures from late winter to early spring result in earlier initiation of cambial reactivation and xylem differentiation in tree stems and an extended growth period. However, earlier cambial reactivation increases the risk for frost damage because the cold tolerance of cambium decreases after cambial reactivation. The present review focuses on temperature regulation on the timing of cambial reactivation and xylem differentiation in trees, and also highlights recent advances in our understanding of seasonal changes in the cold stability of microtubules in trees. The review also summarizes the present understanding of the relationships between the timing of cambial reactivation, the start of xylem differentiation and changes in levels of storage materials in trees, as well as an attempt to identify the source of energy for cell division and differentiation. A better understanding of the mechanisms that regulate wood formation in trees and the influence of environmental conditions on such mechanisms should help in efforts to improve and enhance the exploitation of wood for commercial applications and to prepare for climatic change.

A rapid decrease in temperature induces latewood formation in artificially reactivated cambium of conifer stems.

BACKGROUND AND AIMS: Latewood formation in conifers occurs during the later part of the growing season, when the cell division activity of the cambium declines. Changes in temperature might be important for wood formation in trees. Therefore, the effects of a rapid decrease in temperature on cellular morphology of tracheids were investigated in localized heating-induced cambial reactivation in Cryptomeria japonica trees and in Abies firma seedlings. METHODS: Electric heating tape and heating ribbon were wrapped on the stems of C. japonica trees and A. firma seedlings. Heating was discontinued when 11 or 12 and eight or nine radial files of differentiating and differentiated tracheids had been produced in C. japonica and A. firma stems, respectively. Tracheid diameter, cell wall thickness, percentage of cell wall area and percentage of lumen area were determined by image analysis of transverse sections and scanning electron microscopy. KEY RESULTS: Localized heating induced earlier cambial reactivation and xylem differentiation in stems of C. japonica and A. firma as compared with non-heated stems. One week after cessation of heating, there were no obvious changes in the dimensions of the differentiating tracheids in the samples from adult C. japonica. In contrast, tracheids with a smaller diameter were observed in A. firma seedlings after 1 week of cessation of heating. Two or three weeks after cessation of heating, tracheids with reduced diameters and thickened cell walls were found. The results showed that the rapid decrease in temperature produced slender tracheids with obvious thickening of cell walls that resembled latewood cells. CONCLUSIONS: The results suggest that a localized decrease in temperature of stems induces changes in the diameter and cell wall thickness of differentiating tracheids, indicating that cambium and its derivatives can respond directly to changes in temperature.

Gibberellin is required for the formation of tension wood and stem gravitropism in Acacia mangium seedlings.

BACKGROUND AND AIMS: Angiosperm trees generally form tension wood on the upper sides of leaning stems. The formation of tension wood is an important response to gravitational stimulus. Gibberellin appears to be involved in the differentiation of secondary xylem, but it remains unclear whether gibberellin plays a key role in the formation of tension wood and plant gravitropism. Therefore, a study was designed to investigate the effects of gibberellin and of inhibitors of the synthesis of gibberellin, namely paclobutrazole and uniconazole-P, on the formation of tension wood and negative stem gravitropism in Acacia mangium seedlings. METHODS: Gibberellic acid (GA(3)), paclobutrazole and uniconazole-P were applied to seedlings via the soil in which they were growing. Distilled water was applied similarly as a control. Three days after such treatment, seedlings were tilted at an angle of 45° from the vertical, and samples of stems were collected for analysis 2 weeks, 2 months and 6 months after tilting. The effects of treatments on the stem recovery degree (Rº) were analysed as an index of the negative gravitropism of seedlings, together the width of the region of tension wood in the upper part of inclined stems. KEY RESULTS: It was found that GA(3) stimulated the negative gravitropism of tilted seedling stems of A. mangium, while paclobutrazole and uniconazole-P inhibited recovery to vertical growth. Moreover, GA(3) stimulated the formation of tension wood in tilted A. mangium seedlings, while paclobutrazole and uniconazole-P strongly suppressed the formation of tension wood, as assessed 2 weeks after tilting. CONCLUSIONS: The results suggest that gibberellin plays an important role at the initial stages of formation of tension wood and in stem gravitropism in A. mangium seedlings in response to a gravitational stimulus.

DWARF50 (D50), a rice (Oryza sativa L.) gene encoding inositol polyphosphate 5-phosphatase, is required for proper development of intercalary meristem.

Rice internodes are vital for supporting high-yield panicles, which are controlled by various factors such as cell division, cell elongation and cell wall biosynthesis. Therefore, formation and regulation of the internode cell-producing intercalary meristem (IM) are important for determining the shape of internodes. To understand the regulation of internode development, we analysed a rice dwarf mutant, dwarf 50 (d50). Previously, we reported that parenchyma cells in the elongated internodes of d50 ectopically deposit cell wall phenolics. In this study, we revealed that D50 encodes putative inositol polyphosphate 5-phosphatase (5PTase), which may be involved in phosphoinositide signalling required for many essential cellular functions, such as cytoskeleton organization, endocytosis and vesicular trafficking in eukaryotes. Analysis of the rice genome revealed 20 putative 5PTases including D50. The d50 mutation induced abnormally oriented cell division, irregular deposition of cell wall pectins and thick actin bundles in the parenchyma cells of the IM, resulting in abnormally organized cell files of the internode parenchyma and dwarf phenotype. Our results suggest that the putative 5PTase, encoded by D50, is essential for IM formation, including the direction of cell division, deposition of cell wall pectins and control of actin organization.

Cold stability of microtubules in wood-forming tissues of conifers during seasons of active and dormant cambium.

The cold stability of microtubules during seasons of active and dormant cambium was analyzed in the conifers Abies firma, Abies sachalinensis and Larix leptolepis by immunofluorescence microscopy. Samples were fixed at room temperature and at a low temperature of 2-3°C to examine the effects of low temperature on the stability of microtubules. Microtubules were visible in cambium, xylem cells and phloem cells after fixation at room temperature during seasons of active and dormant cambium. By contrast, fixation at low temperature depolymerized microtubules in cambial cells, differentiating tracheids, differentiating xylem ray parenchyma and phloem ray parenchyma cells during the active season. However, similar fixation did not depolymerize microtubules during cambial dormancy in winter. Our results indicate that the stability of microtubules in cambial cells and cambial derivatives at low temperature differs between seasons of active and dormant cambium. Moreover, the change in the stability of microtubules that we observed at low temperature might be closely related to seasonal changes in the cold tolerance of conifers. In addition, low-temperature fixation depolymerized microtubules in cambial cells and differentiating cells that had thin primary cell walls, while such low-temperature fixation did not depolymerize microtubules in differentiating secondary xylem ray parenchyma cells and tracheids that had thick secondary cell walls. The stability of microtubules at low temperature appears to depend on the structure of the cell wall, namely, primary or secondary. Therefore, we propose that the secondary cell wall might be responsible for the cold stability of microtubules in differentiating secondary xylem cells of conifers.

Natural product-like macrocyclic N-methyl-peptide inhibitors against a ubiquitin ligase uncovered from a ribosome-expressed de novo library.

Naturally occurring peptides often possess macrocyclic and N-methylated backbone. These features grant them structural rigidity, high affinity to targets, proteolytic resistance, and occasionally membrane permeability. Because such peptides are produced by either nonribosomal peptide synthetases or enzymatic posttranslational modifications, it is yet a formidable challenge in degenerating sequence or length and preparing libraries for screening bioactive molecules. Here, we report a new means of synthesizing a de novo library of "natural product-like" macrocyclic N-methyl-peptides using translation machinery under the reprogrammed genetic code, which is coupled with an in vitro display technique, referred to as RaPID (random nonstandard peptides integrated discovery) system. This system allows for rapid selection of strong binders against an arbitrarily chosen therapeutic target. Here, we have demonstrated the selection of anti-E6AP macrocyclic N-methyl-peptides, one of which strongly inhibits polyubiqutination of proteins such as p53.

A flexizyme that selectively charges amino acids activated by a water-friendly leaving group.

We have developed a new flexizyme (a flexible de novo tRNA acylation ribozyme) system, a pair of amino-derivatized benzyl thioester (ABT) and amino flexizyme (aFx). ABT bearing the ammonium ion was designed to render the acyl-donor substrates better water solubility. Although the previously reported flexizymes (eFx and dFx) did not show acylation activity for the ABT derivatives, a new flexizyme variant aFx, generated by in vitro selection against an amino acid activated ABT, exhibits high selectivity toward those activated ABT. The flexizymes system including aFx, eFx, and dFx enables us to prepare a wide variety of acyl-tRNAs charged with non-proteinogenic amino acids.