Clinical characteristics of and growth hormone treatment effects on short stature with type 1 insulin-like growth factor receptor (IGF1R) gene alteration.

Short stature with IGF-1 receptor (IGF1R) gene alteration is known as small-for-gestational-age (SGA) short stature with elevated serum IGF1 levels. Its prevalence and clinical characteristics remain unclear. No adapted treatment is available for short stature related to IGF1R gene alteration in Japan, and genetic testing is not yet widely accessible. We investigated short stature with IGF1R gene alterations and analyzed the clinical data of 13 patients using the results of questionnaires issued to the Japanese Society for Pediatric Endocrinology. Four cases were caused by a deletion of chromosome 15q26.3, and eight were caused by heterozygous pathogenic variants in the IGF1R gene. Cases with deletions showed a more severe degree of growth impairment (-4.5 ± 0.43 SD) than those caused by pathological variants (-2.71 ± 0.15 SD) and were accompanied by neurodevelopmental delay. However, cases caused by pathological variants lacked distinctive features. Only three of the 12 cases demonstrated serum IGF1 values exceeding +2 SD, and the other three had values below 0 SD. Four patients did not meet the criteria for SGA at birth. Six patients received GH therapy for SGA short stature and showed improvement in growth rate without any side effects or elevated serum IGF1 levels during treatment. Elevated IGF1 levels (over +2 SD) after GH treatment should be considered a suspicious finding. Owing to the lack of distinctive features, there was a possibility of undiagnosed cases of this condition. Promoting genetic testing and clinical trials on GH administration for this condition is recommended.

Genetic engineering of a thermophilic acetogen, Moorella thermoacetica Y72, to enable acetoin production.

Acetogens are among the key microorganisms involved in the bioproduction of commodity chemicals from diverse carbon resources, such as biomass and waste gas. Thermophilic acetogens are particularly attractive because fermentation at higher temperatures offers multiple advantages. However, the main target product is acetic acid. Therefore, it is necessary to reshape metabolism using genetic engineering to produce the desired chemicals with varied carbon lengths. Although such metabolic engineering has been hampered by the difficulty involved in genetic modification, a model thermophilic acetogen, M. thermoacetica ATCC 39073, is the case with a few successful cases of C2 and C3 compound production, other than acetate. This brief report attempts to expand the product spectrum to include C4 compounds by using strain Y72 of Moorella thermoacetica. Strain Y72 is a strain related to the type strain ATCC 39073 and has been reported to have a less stringent restriction-modification system, which could alleviate the cumbersome transformation process. A simplified procedure successfully introduced a key enzyme for acetoin (a C4 chemical) production, and the resulting strains produced acetoin from sugars and gaseous substrates. The culture profile revealed varied acetoin yields depending on the type of substrate and culture conditions, implying the need for further engineering in the future. Thus, the use of a user-friendly chassis could benefit the genetic engineering of M. thermoacetica.

Self-Inclusion Complexation of Electron-Accepting Guest into Electron-Donating Cyclic Host by Photoexcitation.

Self-inclusion complexes consisting of host-guest conjugates are one of the unique supramolecular structures because they form in-state and out-state depending on the external stimuli. Despite many reports of the stimuli-responsive self-inclusion complex formation, study of the structural relaxation from out-state to in-state by photoexcitation has been unexplored. Herein, we report that an electron-donating host and an electron-accepting guest conjugate exhibits the structural relaxation from out-state to in-state by photoexcitation. Formation of the in-state in the excited state resulted in exciplex emission along with the locally excited emission from the out-state. Moreover, this structural relaxation by photoexcitation was suppressed not only by temperature, but also by the presence of guest molecules, resulting in changes in the ratio of the dual emission intensities.

Exciplex Formation by Complexation of an Electron-Accepting Guest in an Electron-Donating Pillar[5]arene Host Liquid.

We present a novel system, a liquid-state pillar[5]arene decorated with tri(ethylene oxide) chains, that brings electron-donor and electron-acceptor molecules into proximity for efficient exciplex formation. The electron-accepting guests exhibit a blue-purple emission from a localized excited state upon excitation in common solvents. However, directly dissolving the guests in the electron-donating pillar[5]arene liquid (a bulk system) results in visible green emission from the formed exciplexes. In the bulk system, the guest molecules are always surrounded by excess pillar[5]arene molecules, resulting in the formation of mainly inclusion-type exciplexes. In the bulk system, energy migration occurs between the pillar[5]arene molecules. Excitation of the pillar[5]arenes results in a more intense green exciplex emission than that observed upon direct excitation of the guests. In summary, the pillar[5]arene liquid is a novel system for achieving efficient exciplex formation and energy migration that is different from typical solvent and solid systems.

Isopropanol production via the thermophilic bioconversion of sugars and syngas using metabolically engineered Moorella thermoacetica.

BACKGROUND: Isopropanol (IPA) is a commodity chemical used as a solvent or raw material for polymeric products, such as plastics. Currently, IPA production depends largely on high-CO2-emission petrochemical methods that are not sustainable. Therefore, alternative low-CO2 emission methods are required. IPA bioproduction using biomass or waste gas is a promising method. RESULTS: Moorella thermoacetica, a thermophilic acetogenic microorganism, was genetically engineered to produce IPA. A metabolic pathway related to acetone reduction was selected, and acetone conversion to IPA was achieved via the heterologous expression of secondary alcohol dehydrogenase (sadh) in the thermophilic bacterium. sadh-expressing strains were combined with acetone-producing strains, to obtain an IPA-producing strain. The strain produced IPA as a major product using hexose and pentose sugars as substrates (81% mol-IPA/mol-sugar). Furthermore, IPA was produced from CO, whereas acetate was an abundant byproduct. Fermentation using syngas containing both CO and H2 resulted in higher IPA production at the specific rate of 0.03 h-1. The supply of reducing power for acetone conversion from the gaseous substrates was examined by supplementing acetone to the culture, and the continuous and rapid conversion of acetone to IPA showed a sufficient supply of NADPH for Sadh. CONCLUSIONS: The successful engineering of M. thermoacetica resulted in high IPA production from sugars. M. thermoacetica metabolism showed a high capacity for acetone conversion to IPA in the gaseous substrates, indicating acetone production as the bottleneck in IPA production for further improving the strain. This study provides a platform for IPA production via the metabolic engineering of thermophilic acetogens.

Effects of n-butanol production on metabolism and the photosystem in Synecococcus elongatus PCC 7942 based on metabolic flux and target proteome analyses.

Although n-butanol (BuOH) is an ideal fuel because of its superior physical properties, it has toxicity to microbes. Previously, a Synechococcus elongatus PCC 7942 derivative strain that produces BuOH from CO2 was developed by introducing six heterologous genes (BUOH-SE strain). To identify the bottleneck in BuOH production, the effects of BuOH production and its toxicity on central metabolism and the photosystem were investigated. Parental (WT) and BUOH-SE strains were cultured under autotrophic conditions. Consistent with the results of a previous study, BuOH production was observed only in the BUOH-SE strain. Isotopically non-stationary 13C-metabolic flux analysis revealed that the CO2 fixation rate was much larger than the BuOH production rate in the BUOH-SE strain (1.70 vs 0.03 mmol gDCW-1 h-1), implying that the carbon flow for BuOH biosynthesis was less affected by the entire flux distribution. No large difference was observed in the flux of metabolism between the WT and BUOH-SE strains. Contrastingly, in the photosystem, the chlorophyll content and maximum O2 evolution rate per dry cell weight of the BUOH-SE strain were decreased to 81% and 43% of the WT strain, respectively. Target proteome analysis revealed that the amounts of some proteins related to antennae (ApcA, ApcD, ApcE, and CpcC), photosystem II (PsbB, PsbU, and Psb28-2), and cytochrome b6f complex (PetB and PetC) in photosystems decreased in the BUOH-SE strain. The activation of photosynthesis would be a novel approach for further enhancing BuOH production in S. elongatus PCC 7942.

Diastereoselective Rotaxane Synthesis with Pillar[5]arenes via Co-crystallization and Solid-State Mechanochemical Processes.

Chiral rotaxanes have attracted much attention in recent decades for their unique chirality based on their interlocked structures. Thus, selective synthesis methods of chiral rotaxanes have been developed. The introduction of substituents with chiral centers to produce diastereomers is a powerful strategy for the construction of chiral rotaxanes. However, in case of a small energy difference between the diastereomers, diastereoselective synthesis is extremely difficult. Herein, we report a new diastereoselective rotaxane synthesis method using solid-phase diastereoselective [3]pseudorotaxane formation and mechanochemical solid-phase end-capping reactions of the [3]pseudorotaxanes. By co-crystallization of stereodynamic planar chiral pillar[5]arene with stereogenic carbons at both rims and axles with suitable end groups and lengths, the [3]pseudorotaxane with a high diastereomeric excess (ca. 92% de) was generated in the solid state because of higher effective molarity with aid by packing effects and significant energy differences between [3]pseudorotaxane diastereomers. In contrast, the de of the pillar[5]arene was low in solution (ca. 10% de) because of a small energy difference between diastereomers. Subsequent end-capping reactions of the polycrystalline [3]pseudorotaxane with high de in solvent-free conditions successfully yielded rotaxanes while maintaining the high de generated by the co-crystallization.

Extracellular Vesicles and Cx43-Gap Junction Channels Are the Main Routes for Mitochondrial Transfer from Ultra-Purified Mesenchymal Stem Cells, RECs.

Mitochondria are essential organelles for maintaining intracellular homeostasis. Their dysfunction can directly or indirectly affect cell functioning and is linked to multiple diseases. Donation of exogenous mitochondria is potentially a viable therapeutic strategy. For this, selecting appropriate donors of exogenous mitochondria is critical. We previously demonstrated that ultra-purified bone marrow-derived mesenchymal stem cells (RECs) have better stem cell properties and homogeneity than conventionally cultured bone marrow-derived mesenchymal stem cells. Here, we explored the effect of contact and noncontact systems on three possible mitochondrial transfer mechanisms involving tunneling nanotubes, connexin 43 (Cx43)-mediated gap junction channels (GJCs), and extracellular vesicles (Evs). We show that Evs and Cx43-GJCs provide the main mechanism for mitochondrial transfer from RECs. Through these two critical mitochondrial transfer pathways, RECs could transfer a greater number of mitochondria into mitochondria-deficient (ρ0) cells and could significantly restore mitochondrial functional parameters. Furthermore, we analyzed the effect of exosomes (EXO) on the rate of mitochondrial transfer from RECs and recovery of mitochondrial function. REC-derived EXO appeared to promote mitochondrial transfer and slightly improve the recovery of mtDNA content and oxidative phosphorylation in ρ0 cells. Thus, ultrapure, homogenous, and safe stem cell RECs could provide a potential therapeutic tool for diseases associated with mitochondrial dysfunction.

Efficient synthesis and unit-selective π-extension of π-fused [4.3.3]propellane as a chiral building block.

A three-dimensional skeleton, π-fused [4.3.3]propellane, was constructed and derivatized by selective π-extension at the two naphthalene units. The obtained propellanes existed as stereoisomers different in spatial arrangement, one of which displayed a chiroptical response originating from through-space interactions between 5-azachrysenes in a skew position.

Enhancing acetone production from H2 and CO2 using supplemental electron acceptors in an engineered Moorella thermoacetica.

Acetogens grow autotrophically and use hydrogen (H2) as the energy source to fix carbon dioxide (CO2). This feature can be applied to gas fermentation, contributing to a circular economy. A challenge is the gain of cellular energy from H2 oxidation, which is substantially low, especially when acetate formation coupled with ATP production is diverted to other chemicals in engineered strains. Indeed, an engineered strain of the thermophilic acetogen Moorella thermoacetica that produces acetone lost autotrophic growth on H2 and CO2. We aimed to recover autotrophic growth and enhance acetone production, in which ATP production was assumed to be a limiting factor, by supplementing with electron acceptors. Among the four selected electron acceptors, thiosulfate and dimethyl sulfoxide (DMSO) enhanced both bacterial growth and acetone titers. DMSO was the most effective and was further analyzed. We showed that DMSO supplementation enhanced intracellular ATP levels, leading to increased acetone production. Although DMSO is an organic compound, it functions as an electron acceptor, not a carbon source. Thus, supplying electron acceptors is a potential strategy to complement the low ATP production caused by metabolic engineering and to improve chemical production from H2 and CO2.

Adaptive Planar Chirality of Pillar[5]arenes Invertible by n-Alkane Lengths.

Chirality of host molecules can be induced and/or inverted by the guest molecules. However, the adapting chirality of hosts to the length of n-alkanes remains a great challenge because n-alkanes are neutral, achiral, and linear molecules, resulting in a weak interaction with most compounds. Herein, we report a system with chirality adapted to n-alkane lengths, using a pillar[5]arene-based macrocyclic host, S-Br, which contains five stereogenic carbons and five terminal bromine atoms on each rim. The electron-rich cavity of S-Br could include n-alkanes and the planar-chiral isomers sensitively inverted in response to the lengths of the complexed n-alkanes. The inclusion of a short n-alkane such as n-pentane made S-Br more inclined to be in the pS-form, whereas the inclusion of long n-alkanes such as n-heptane made the pR-form more favorable. The difference in the stability of the isomers was supported by the crystal structures and the theoretical calculations. Furthermore, temperature drives the adaptive chirality of S-Br with n-alkanes. An n-alkane with middle length, n-hexane, showed the dominance of the pR-form of S-Br at a higher temperature, whereas the pS-form was shown at a lower temperature.

Dynamic-to-Static Planar Chirality Conversion in Pillar[5]arenes Regulated by Guest Solvents or Amplified by Crystallization.

Controlling dynamic chirality and memorizing the controlled chirality are important. Chirality memory has mainly been achieved using noncovalent interactions. However, in many cases, the memorized chirality arising from noncovalent interactions is erased by changing the conditions such as the solvent and temperature. In this study, the dynamic planar chirality of pillar[5]arenes was successfully converted into static planar chirality by introducing bulky groups through covalent bonds. Before introducing the bulky groups, pillar[5]arene with stereogenic carbon atoms at both rims existed as a pair of diastereomers, and thus showed planar chiral inversion that was dependent on the chain length of the guest solvent. The pS and pR forms, regulated by guest solvents, were both diastereomerically memorized by introducing bulky groups. Furthermore, the diastereomeric excess was amplified by crystallization of the pillar[5]arene. The subsequent introduction of bulky groups yielded pillar[5]arene with an excellent diastereomeric excess (95 % de).

Highly-purified rapidly expanding clones, RECs, are superior for functional-mitochondrial transfer.

BACKGROUND: Mitochondrial dysfunction caused by mutations in mitochondrial DNA (mtDNA) or nuclear DNA, which codes for mitochondrial components, are known to be associated with various genetic and congenital disorders. These mitochondrial disorders not only impair energy production but also affect mitochondrial functions and have no effective treatment. Mesenchymal stem cells (MSCs) are known to migrate to damaged sites and carry out mitochondrial transfer. MSCs grown using conventional culture methods exhibit heterogeneous cellular characteristics. In contrast, highly purified MSCs, namely the rapidly expanding clones (RECs) isolated by single-cell sorting, display uniform MSCs functionality. Therefore, we examined the differences between RECs and MSCs to assess the efficacy of mitochondrial transfer. METHODS: We established mitochondria-deficient cell lines (ρ0 A549 and ρ0 HeLa cell lines) using ethidium bromide. Mitochondrial transfer from RECs/MSCs to ρ0 cells was confirmed by PCR and flow cytometry analysis. We examined several mitochondrial functions including ATP, reactive oxygen species, mitochondrial membrane potential, and oxygen consumption rate (OCR). The route of mitochondrial transfer was identified using inhibition assays for microtubules/tunneling nanotubes, gap junctions, or microvesicles using transwell assay and molecular inhibitors. RESULTS: Co-culture of ρ0 cells with MSCs or RECs led to restoration of the mtDNA content. RECs transferred more mitochondria to ρ0 cells compared to that by MSCs. The recovery of mitochondrial function, including ATP, OCR, mitochondrial membrane potential, and mitochondrial swelling in ρ0 cells co-cultured with RECs was superior than that in cells co-cultured with MSCs. Inhibition assays for each pathway revealed that RECs were sensitive to endocytosis inhibitor, dynasore. CONCLUSIONS: RECs might serve as a potential therapeutic strategy for diseases linked to mitochondrial dysfunction by donating healthy mitochondria.

Real-time chirality transfer monitoring from statistically random to discrete homochiral nanotubes.

Real time monitoring of chirality transfer processes is necessary to better understand their kinetic properties. Herein, we monitor an ideal chirality transfer process from a statistically random distribution to a diastereomerically pure assembly in real time. The chirality transfer is based on discrete trimeric tubular assemblies of planar chiral pillar[5]arenes, achieving the construction of diastereomerically pure trimers of pillar[5]arenes through synergistic effect of ion pairing between a racemic rim-differentiated pillar[5]arene pentaacid bearing five benzoic acids on one rim and five alkyl chains on the other, and an optically resolved pillar[5]arene decaamine bearing ten amines. When the decaamine is mixed with the pentaacid, the decaamine is sandwiched by two pentaacids through ten ion pairs, initially producing a statistically random mixture of a homochiral trimer and two heterochiral trimers. The heterochiral trimers gradually dissociate and reassemble into the homochiral trimers after unit flipping of the pentaacid, leading to chirality transfer from the decaamine and producing diastereomerically pure trimers.

Superior Multielectron-Transferring Energy Storage by π-d Conjugated Frameworks.

Reversible multielectron-transfer materials are of considerable interest because of the potential impact to advance present electrochemical energy storage technology by boosting energy density. To date, a few oxide-based materials can reach an electron-transfer number per metal-cation (eM ) larger than 2 upon a (de)intercalation mechanism. However, these materials suffer from degradation due to irreversible rearrangements of the cation-oxygen bonds, and are based on precious metals, for example, Ir and Ru. Hence, a design of the non-oxide-based reversible multielectron-transfer materials with abundant elements can provide a promising alternative. Herein, it is demonstrated that the bis(diimino)copper framework can show eM  = 3.5 with cation/anion co-redox mechanism together with a dual-ion mechanism. In this study, the role of the cation-anion interactions is unveiled by using an experiment/theory collaboration applied to a series of the model non-oxide abundant electrode systems based on different metal-nitrogen bonds. These models provide designer multielectron-transfer due to the tunable π-d conjugated electronic structures. It is found that the Cu-nitrogen bonds show a unique reversible rearrangement upon Li-intercalation, and this process responds to acquire a significant reversible multielectron-transfer. This work provides new insights into the affordable multielectron-transfer electrodes and uncovers an alternative strategy to advance the electrochemical energy storage reactions.

CPL on/off control of an assembled system by water soluble macrocyclic chiral sources with planar chirality.

Herein, we report the synthesis and planar chiral properties of a pair of water-soluble cationic pillar[5]arenes with stereogenic carbons. Interestingly, although units of the molecules were rotatable, only one planar chiral diastereomer existed in water in both cases. As a new type of chiral source, these molecules transmitted chiral information from the planar chiral cavities to the assembly of a water-soluble extended π-conjugated compound, affording circularly polarized luminescence (CPL). The chirality transfer process and resulting CPL were extremely sensitive to the feed ratio of the chiral pillar[5]arenes owing to the combined action of their planar chirality, bulkiness, and strong binding properties. When a limited amount of chiral source was added, further assembly of the extended π-conjugated compound into helical fibers with CPL was triggered. Unexpectedly, larger amounts of chiral source destroyed the helical fiber assemblies, resulting in elimination of the chirality and CPL properties from the assembled structures.

Reversible Hydrogenase Activity Confers Flexibility to Balance Intracellular Redox in Moorella thermoacetica.

Hydrogen (H2) converted to reducing equivalents is used by acetogens to fix and metabolize carbon dioxide (CO2) to acetate. The utilization of H2 enables not only autotrophic growth, but also mixotrophic metabolism in acetogens, enhancing carbon utilization. This feature seems useful, especially when the carbon utilization efficiency of organic carbon sources is lowered by metabolic engineering to produce reduced chemicals, such as ethanol. The potential advantage was tested using engineered strains of Moorella thermoacetica that produce ethanol. By adding H2 to the fructose-supplied culture, the engineered strains produced increased levels of acetate, and a slight increase in ethanol was observed. The utilization of a knockout strain of the major acetate production pathway, aimed at increasing the carbon flux to ethanol, was unexpectedly hindered by H2-mediated growth inhibition in a dose-dependent manner. Metabolomic analysis showed a significant increase in intracellular NADH levels due to H2 in the ethanol-producing strain. Higher NADH level was shown to be the cause of growth inhibition because the decrease in NADH level by dimethyl sulfoxide (DMSO) reduction recovered the growth. When H2 was not supplemented, the intracellular NADH level was balanced by the reversible electron transfer from NADH oxidation to H2 production in the ethanol-producing strain. Therefore, reversible hydrogenase activity confers the ability and flexibility to balance the intracellular redox state of M. thermoacetica. Tuning of the redox balance is required in order to benefit from H2-supplemented mixotrophy, which was confirmed by engineering to produce acetone.

Metabolomic Evaluation of the Central Metabolic Pathways of Mannosylerythritol Lipid Biosynthesis in Moesziomyces antarcticus T-34.

Moesziomyces antarcticus is a basidiomycetous yeast that produces mannosylerythritol lipids (MELs), which have potential applications as bio-based functional materials in various oleochemical industries, the cosmetics, toiletry, agriculture, and pharmaceutical industries. To better understand the MEL producer, we characterized the central metabolic pathways of M. antarcticus strain T-34 grown on glucose or olive oil via metabolomics. The relative fatty acid content was higher in the cells cultured in olive oil compared to glucose, while the acetyl-CoA content was lower in cells cultured in olive oil. The levels of the tricarboxylic acid cycle metabolites citrate/isocitrate, α-ketoglutarate, and succinate were lower in olive oil compared to glucose, while fumarate and malate levels exhibited the opposite pattern. Pyruvate was not detected in olive oil compared to glucose culture. The levels of glycerol, as well as trehalose, myo-inositol, threitol/erythritol, and mannitol/sorbitol, were higher in olive oil compared to glucose cultures. The ATP level was lower in olive oil compared to glucose culture, although the assimilation of fatty acids produced by digestion of olive oil should promote large amounts of ATP production. The possibility that ATP regeneration by respiratory chain complex promote oil utilization and MEL production in M. antarcticus T-34 was found based on the results of this metabolomic analysis.

Draft Genome Sequence of a Basidiomycetous Yeast, Ustilago shanxiensis CBS 10075, Which Produces Mannosylerythritol Lipids.

The basidiomycetous yeast Ustilago shanxiensis CBS 10075, which was isolated from a wilting leaf in China, produces mannosylerythritol lipid (MEL) biosurfactants. Here, we report the draft genome sequence of U. shanxiensis CBS 10075, which was 21.7 Mbp in size, with a GC content of 52.55%, comprising 65 scaffolds.