Efficient pathway-driven scyllo-inositol production from myo-inositol using thermophilic cells and mesophilic inositol dehydrogenases: a novel strategy for pathway control.

Mesophilic enzymes, which are active at moderate temperatures, may dominate enzymatic reactions even in the presence of thermophilic crude enzymes. This study was conducted to investigate this hypothesis with mesophilic inositol dehydrogenases (IolG and IolX) produced in Geobacillus kaustophilus HTA426. To ensure the efficient production of mesophilic enzymes, we first screened for promoters induced at moderate temperatures using transcriptome analysis and identified four genes highly expressed at 30°C in the thermophile. We further characterized these promoters using fluorescent reporter assays to determine that the mti3 promoter could direct efficient gene expression at 40°C. We cloned the promoter into an Escherichia coli-Geobacillus shuttle plasmid and confirmed that the resulting vector functioned in G. kaustophilus and other thermophiles. We then used this vector for the cooperative expression of the iolG and iolX genes from Bacillus subtilis 168. G. kaustophilus cells carrying the expression vector were incubated at 60°C for cellular propagation and then at 40°C for the production of IolG and IolX. When the cells were permeabilized, IolG and IolX acted as catalysts to convert exogenous myo-inositol into scyllo-inositol at 30°C. In a scaled-up reaction, 10 g of myo-inositol was converted to 1.8 g of scyllo-inositol, which was further purified to yield 970 mg of pure powder. Notably, myo-inositol was degraded by intrinsic enzymes of G. kaustophilus at 60°C but not at 30°C, supporting our initial hypothesis. We indicate that this approach is useful for preparing enzyme cocktails without the need for purification. IMPORTANCE: Enzyme cocktails are commonly employed for cell-free chemical synthesis; however, their preparation involves cumbersome processes. This study affirms that mesophilic enzymes in thermophilic crude extracts can function as specific catalysts at moderate temperatures, akin to enzyme cocktails. The catalyst was prepared by permeabilizing cells without the need for concentration, extraction, or purification processes; hence, its preparation was considerably simpler compared with conventional methods for enzyme cocktails. This approach was employed to produce pure scyllo-inositol from an economical substrate. Notably, this marks the first large-scale preparation of pure scyllo-inositol, holding potential pharmaceutical significance as scyllo-inositol serves as a promising agent for certain diseases but is currently expensive. Moreover, this approach holds promise for application in pathway engineering within living cells. The envisioned pathway is designed without chromosomal modification and is simply regulated by switching culture temperatures. Consequently, this study introduces a novel platform for both whole-cell and cell-free synthetic systems.

A highly specific antibody against the core fucose of the N-glycan in IgG identifies the pulmonary diseases and its regulation by CCL2.

Glycan structure is often modulated in disease or predisease states, suggesting that such changes might serve as biomarkers. Here, we generated a monoclonal antibody (mAb) against the core fucose of the N-glycan in human IgG. Notably, this mAb can be used in Western blotting and ELISA. ELISA using this mAb revealed a low level of the core fucose of the N-glycan in IgG, suggesting that the level of acore fucosylated (noncore fucosylated) IgG was increased in the sera of the patients with lung cancer, chronic obstructive pulmonary disease, and interstitial pneumonia compared to healthy subjects. In a coculture analysis using human lung adenocarcinoma A549 cells and antibody-secreting B cells, the downregulation of the FUT8 (α1,6 fucosyltransferase) gene and a low level of core fucose of the N-glycan in IgG in antibody-secreting B cells were observed after coculture. A dramatic alteration in gene expression profiles for cytokines, chemokines, and their receptors were also observed after coculturing, and we found that the identified C-C motif chemokine 2 was partially involved in the downregulation of the FUT8 gene and the low level of core fucose of the N-glycan in IgG in antibody-secreting B cells. We also developed a latex turbidimetric immunoassay using this mAb. These results suggest that communication with C-C motif chemokine 2 between lung cells and antibody-secreting B cells downregulate the level of core fucose of the N-glycan in IgG, i.e., the increased level of acore fucosylated (noncore fucosylated) IgG, which would be a novel biomarker for the diagnosis of patients with pulmonary diseases.

Flexible Coordination of Flexible Limbs: Decentralized Control Scheme for Inter- and Intra-Limb Coordination in Brittle Stars' Locomotion.

Conventional mobile robots have difficulties adapting to unpredictable environments or performing adequately after undergoing physical damages in realtime operation, unlike animals. We address this issue by focusing on brittle stars, an echinoderm related to starfish. Most brittle stars have five flexible arms, and they can coordinate among the arms (i.e., inter-arm coordination) as well as the many bodily degrees of freedom within each arm (i.e., intra-arm coordination). They can move in unpredictable environments while promptly adapting to those, and to their own physical damages (e.g., arm amputation). Our previous work focused on the inter-arm coordination by studying trimmed-arm brittle stars. Herein, we extend our previous work and propose a decentralized control mechanism that enables coupling between the inter-arm and intra-arm coordination. We demonstrate via simulations and real-world experiments with a brittle star-like robot that the behavior of brittle stars when they are intact and undergoing shortening or amputation of arms can be replicated.

Evaluation of Drug Exposure and Metabolism in Locust and Zebrafish Brains Using Mass Spectrometry Imaging.

Studying how and where drugs are metabolized in the brain is challenging. In an entire organism, peripheral metabolism produces many of the same metabolites as those in the brain, and many of these metabolites can cross the blood-brain barrier from the periphery, thus making the relative contributions of hepatic and brain metabolism difficult to study in vivo. In addition, drugs and metabolites contained in ventricles and in the residual blood of capillaries in the brain may overestimate drugs' and metabolites' concentrations in the brain. In this study, we examine locusts and zebrafish using matrix assisted laser desorption ionization mass spectrometry imaging to study brain metabolism and distribution. These animal models are cost-effective and ethically sound for initial drug development studies.

A brittle star-like robot capable of immediately adapting to unexpected physical damage.

A major challenge in robotic design is enabling robots to immediately adapt to unexpected physical damage. However, conventional robots require considerable time (more than several tens of seconds) for adaptation because the process entails high computational costs. To overcome this problem, we focus on a brittle star-a primitive creature with expendable body parts. Brittle stars, most of which have five flexible arms, occasionally lose some of them and promptly coordinate the remaining arms to escape from predators. We adopted a synthetic approach to elucidate the essential mechanism underlying this resilient locomotion. Specifically, based on behavioural experiments involving brittle stars whose arms were amputated in various ways, we inferred the decentralized control mechanism that self-coordinates the arm motions by constructing a simple mathematical model. We implemented this mechanism in a brittle star-like robot and demonstrated that it adapts to unexpected physical damage within a few seconds by automatically coordinating its undamaged arms similar to brittle stars. Through the above-mentioned process, we found that physical interaction between arms plays an essential role for the resilient inter-arm coordination of brittle stars. This finding will help develop resilient robots that can work in inhospitable environments. Further, it provides insights into the essential mechanism of resilient coordinated motions characteristic of animal locomotion.

Generation of tetracycline-inducible conditional gene knockout cells in a human Nalm-6 cell line.

Conditional gene knockout by homologous recombination combined with an inducible gene expression system is a powerful approach for studying gene function, although homologous recombination in human cells occurs infrequently. The tetracycline-regulated gene expression (Tet-Off) system is a convenient method for achieving conditional gene knockout, but it is not always promising in Nalm-6, a rare human cell line highly effective for gene targeting. Here we modified the Tet-Off system and applied it to the Nalm-6 cell line successfully by using an internal ribosome entry site to drive a selectable marker from the same tetracycline-responsive promoter for the transgene. We also inserted the gene for the tetracycline-controlled transactivator under the control of a potent CAG promoter. These modifications enabled us to easily obtain rare clones that express optimal amounts of tetracycline-regulated transgenes. We thereby generated a 'tetracycline-inducible conditional gene knockout' for the proliferation-associated SNF2-like gene (PASG) in a Nalm-6 cell line, in which the expression of PASG can be depleted in a tetracycline-dependent manner on a knockout background. This method is applicable to any human genes, making this gene-targeting system using the Nalm-6 cell line a promising tool for analyzing gene function.

Research on radiation protection in the application of new technologies for proton and heavy ion radiotherapy.

Particle radiotherapy using proton and heavy ion beams has shown improved clinical results and is a promising cancer therapy which is expected to gradually spread in Japan. There are, however, no special regulations for radiotherapy treatment facilities. They have been operated under the same safety regulations as for a research facility using a research accelerator. Significantly high-energy radiation is necessary for particle radiotherapy compared with conventional radiation therapy. The treatment facility, therefore, should have a large accelerator, which is installed in a room with a thick shield wall. Data on radiation protection for such high energy medical facilities is fragmentary and insufficient. In this study, we examined the necessity of other regulations for the safe operation of medical facilities for particle radiotherapy. First, we measured activation levels of the therapeutic devices and of patients. Next the safety level of the medical facility was evaluated from the viewpoint of radiation protection. We have confirmed the facilities can be safely operated by present regulations given in the Law Concerning Prevention from Radiation Hazards due to Radiation Isotopes, etc. or the Law for Health Protection and Medical Care.

Essential role of chromatin assembly factor-1-mediated rapid nucleosome assembly for DNA replication and cell division in vertebrate cells.

Chromatin assembly factor-1 (CAF-1), a complex consisting of p150, p60, and p48 subunits, is highly conserved from yeast to humans and facilitates nucleosome assembly of newly replicated DNA in vitro. To investigate roles of CAF-1 in vertebrates, we generated two conditional DT40 mutants, respectively, devoid of CAF-1p150 and p60. Depletion of each of these CAF-1 subunits led to delayed S-phase progression concomitant with slow DNA synthesis, followed by accumulation in late S/G2 phase and aberrant mitosis associated with extra centrosomes, and then the final consequence was cell death. We demonstrated that CAF-1 is necessary for rapid nucleosome formation during DNA replication in vivo as well as in vitro. Loss of CAF-1 was not associated with the apparent induction of phosphorylations of S-checkpoint kinases Chk1 and Chk2. To elucidate the precise role of domain(s) in CAF-1p150, functional dissection analyses including rescue assays were preformed. Results showed that the binding abilities of CAF-1p150 with CAF-1p60 and DNA polymerase sliding clamp proliferating cell nuclear antigen (PCNA) but not with heterochromatin protein HP1-gamma are required for cell viability. These observations highlighted the essential role of CAF-1-dependent nucleosome assembly in DNA replication and cell proliferation through its interaction with PCNA.

Histone acetyltransferase 1 is dispensable for replication-coupled chromatin assembly but contributes to recover DNA damages created following replication blockage in vertebrate cells.

Histone acetyltransferase 1 (HAT1) is implicated for diacetylation of Lys-5 and Lys-12 of newly synthesized histone H4, the biological significance of which remains unclear. To investigate the in vivo role of HAT1, we generated HAT1-deficient DT40 clone (HAT1(-/-)). HAT1(-/-) cells exhibited greatly reduced diacetylation levels of Lys-5 and Lys-12, and acetylation level of Lys-5 of cytosolic and chromatin histones H4, respectively. The in vitro nucleosome assembly assay and in vivo MNase digestion assay revealed that HAT1 and diacetylation of Lys-5 and Lys-12 of histone H4 are dispensable for replication-coupled chromatin assembly. HAT1(-/-) cells had mild growth defect, conferring sensitivities to methyl methanesulfonate and camptothecin that enforce replication blocks creating DNA double strand breaks. Such heightened sensitivities were associated with prolonged late-S/G2 phase. These results indicate that HAT1 participates in recovering replication block-mediated DNA damages, probably through chromatin modulation based on acetylation of Lys-5 and Lys-12 of histone H4.

Asf1 is required for viability and chromatin assembly during DNA replication in vertebrate cells.

Asf1 (anti-silencing function 1), a well conserved protein from yeast to humans, acts as a histone chaperone and is predicted to participate in a variety of chromatin-mediated cellular processes. To investigate the physiological role of vertebrate Asf1 in vivo, we generated a conditional Asf1-deficient mutant from chicken DT40 cells. Induction of Asf1 depletion resulted in the accumulation of cells in S phase with decreased DNA replication and increased mitotic aberrancy forming multipolar spindles, leading to cell death. In addition, nascent chromatin in Asf1-depleted cells showed increased nuclease sensitivity, indicating impaired nucleosome assembly during DNA replication. Complementation analyses revealed that the functional domain of Asf1 for cell viability was confined to the N-terminal core domain (amino acids 1-155) that is a binding platform for histones H3/H4, CAF-1p60, and HIRA, whereas Asf1 mutant proteins, abolishing binding abilities with both p60 and HIRA, exhibit no effect on viability. These results together indicate that the vertebrate Asf1 plays a crucial role in replication-coupled chromatin assembly, cell cycle progression, and cellular viability and provide a clue of a possible role in a CAF-1- and HIRA-independent chromatin-modulating process for cell proliferation.

Chromatin assembly factor 1 ensures the stable maintenance of silent chromatin states in Arabidopsis.

Newly synthesized DNA is rapidly assembled into mature nucleosomes by the deposition of pre-existing and nascent histones, and some parts of this process are facilitated by chromatin assembly factor 1 (CAF-1). Loss-of-function mutants of CAF-1 in Arabidopsis, fasciata (fas), show a variety of morphological abnormalities and unique defects in gene expression in the meristems. In order to clarify the implications of CAF-1 in the maintenance of chromatin states in higher eukaryotes, we investigated transcriptional gene silencing (TGS) of various genes in fas mutants. Here, we show that TGS of endogenous CACTA transposons was released in a stochastic manner in fas. Other endogenous silent genes, a transposon AtMu1 and a hypothetical gene T5L23.26 at a heterochromatin knob, were also transcriptionally activated, and the activation of the three different silent loci at different chromosomal sites occurred non-concomitantly with each other. Furthermore, TGS of the silent beta-glucuronidase (GUS) transgene was also de-repressed randomly in fas. We conclude that CAF-1 ensures the stable inheritance of epigenetic states through growth and development in Arabidopsis.

Dominant negative E2F inhibits progression of the cell cycle after the midblastula transition in Xenopus.

The cleavage cycle, which is initiated by fertilization, consists of only S and M phases, and the gap phases (G1 and G2) appear after the midblastula transition (MBT) in the African clawed frog, Xenopus laevis. During early development in Xenopus, we examined the E2F activity, which controls transition from the G1 to S phase in the somatic cell cycle. Gel retardation and transactivation assays revealed that, although the E2F protein was constantly present throughout early development, the E2F transactivation activity was induced in a stage-specific manner, that is, low before MBT and rapidly increased after MBT. Introduction of the recombinant dominant negative E2F (dnE2F), but not the control, protein into the 2-cell stage embryos specifically suppressed E2F activation after MBT. Cells in dnE2F-injected embryos appeared normal before MBT, but ceased to proliferate and eventually died at the gastrula. These cells contained decreased cdk activity with enhanced inhibitory phosphorylation of Cdc2 at Tyr15. Thus, E2F activity is required for cell cycle progression and cell viability after MBT, but not essential for MBT transition and developmental progression during the cleavage stage.