Changes in repair pathways of radiation-induced DNA double-strand breaks at the midblastula transition in Xenopus embryo.

Ionizing radiation (IR) causes DNA damage, particularly DNA double-strand breaks (DSBs), which have significant implications for genome stability. The major pathways of repairing DSBs are homologous recombination (HR) and nonhomologous end joining (NHEJ). However, the repair mechanism of IR-induced DSBs in embryos is not well understood, despite extensive research in somatic cells. The externally developing aquatic organism, Xenopus tropicalis, serves as a valuable model for studying embryo development. A significant increase in zygotic transcription occurs at the midblastula transition (MBT), resulting in a longer cell cycle and asynchronous cell divisions. This study examines the impact of X-ray irradiation on Xenopus embryos before and after the MBT. The findings reveal a heightened X-ray sensitivity in embryos prior to the MBT, indicating a distinct shift in the DNA repair pathway during embryo development. Importantly, we show a transition in the dominant DSB repair pathway from NHEJ to HR before and after the MBT. These results suggest that the MBT plays a crucial role in altering DSB repair mechanisms, thereby influencing the IR sensitivity of developing embryos.

The proofreading exonuclease of leading-strand DNA polymerase epsilon prevents replication fork collapse at broken template strands.

Leading-strand DNA replication by polymerase epsilon (Polϵ) across single-strand breaks (SSBs) causes single-ended double-strand breaks (seDSBs), which are repaired via homology-directed repair (HDR) and suppressed by fork reversal (FR). Although previous studies identified many molecules required for hydroxyurea-induced FR, FR at seDSBs is poorly understood. Here, we identified molecules that specifically mediate FR at seDSBs. Because FR at seDSBs requires poly(ADP ribose)polymerase 1 (PARP1), we hypothesized that seDSB/FR-associated molecules would increase tolerance to camptothecin (CPT) but not the PARP inhibitor olaparib, even though both anti-cancer agents generate seDSBs. Indeed, we uncovered that Polϵ exonuclease and CTF18, a Polϵ cofactor, increased tolerance to CPT but not olaparib. To explore potential functional interactions between Polϵ exonuclease, CTF18, and PARP1, we created exonuclease-deficient POLE1exo-/-, CTF18-/-, PARP1-/-, CTF18-/-/POLE1exo-/-, PARP1-/-/POLE1exo-/-, and CTF18-/-/PARP1-/- cells. Epistasis analysis indicated that Polϵ exonuclease and CTF18 were interdependent and required PARP1 for CPT tolerance. Remarkably, POLE1exo-/- and HDR-deficient BRCA1-/- cells exhibited similar CPT sensitivity. Moreover, combining POLE1exo-/- with BRCA1-/- mutations synergistically increased CPT sensitivity. In conclusion, the newly identified PARP1-CTF18-Polϵ exonuclease axis and HDR act independently to prevent fork collapse at seDSBs. Olaparib inhibits this axis, explaining the pronounced cytotoxic effects of olaparib on HDR-deficient cells.

Repair of topoisomerase 1-induced DNA damage by tyrosyl-DNA phosphodiesterase 2 (TDP2) is dependent on its magnesium binding.

Topoisomerases are enzymes that relax DNA supercoiling during replication and transcription. Camptothecin, a topoisomerase 1 (TOP1) inhibitor, and its analogs trap TOP1 at the 3'-end of DNA as a DNA-bound intermediate, resulting in DNA damage that can kill cells. Drugs with this mechanism of action are widely used to treat cancers. It has previously been shown that tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs TOP1-induced DNA damage generated by camptothecin. In addition, tyrosyl-DNA phosphodiesterase 2 (TDP2) plays critical roles in repairing topoisomerase 2 (TOP2)-induced DNA damage at the 5'-end of DNA and in promoting the repair of TOP1-induced DNA damage in the absence of TDP1. However, the catalytic mechanism by which TDP2 processes TOP1-induced DNA damage has not been elucidated. In this study, we found that a similar catalytic mechanism underlies the repair of TOP1- and TOP2-induced DNA damage by TDP2, with Mg2+-TDP2 binding playing a role in both repair mechanisms. We show chain-terminating nucleoside analogs are incorporated into DNA at the 3'-end and abort DNA replication to kill cells. Furthermore, we found that Mg2+-TDP2 binding also contributes to the repair of incorporated chain-terminating nucleoside analogs. Overall, these findings reveal the role played by Mg2+-TDP2 binding in the repair of both 3'- and 5'-blocking DNA damage.

Amino Acid Conjugates of ent-Beyerane-type Diterpenoids and Oleanane-type Triterpenoids: Synthesis and Evaluation of Cytotoxic Activities.

The aim of this study was to increase the cytotoxic activities of terpenoids via amino acid conjugation. Thus, 21 new ester derivatives (5-15, 19-27, and 29) were prepared by conjugation of the hydroxy groups in ent-beyerane-type diterpenoids (4) and oleanane-type triterpenoids (18), and their cytotoxic activity against three human cancer cell lines (leukemia, lung, and stomach) were evaluated. The prepared compounds showed cytotoxic effects; in particular, all amino acid conjugates of ent-beyerane-type diterpenoids (5-13) exhibited potent cytotoxic activity (IC50 1.0-3.7 µM for HL60, 1.7-8.2 µM for A549, and 2.5-11.7 µM for MKN45). In addition, no differences were observed in the cytotoxic activities of l- and d-type amino acid conjugates.

ATM suppresses c-Myc overexpression in the mammary epithelium in response to estrogen.

ATM gene mutation carriers are predisposed to estrogen-receptor-positive breast cancer (BC). ATM prevents BC oncogenesis by activating p53 in every cell; however, much remains unknown about tissue-specific oncogenesis after ATM loss. Here, we report that ATM controls the early transcriptional response to estrogens. This response depends on topoisomerase II (TOP2), which generates TOP2-DNA double-strand break (DSB) complexes and rejoins the breaks. When TOP2-mediated ligation fails, ATM facilitates DSB repair. After estrogen exposure, TOP2-dependent DSBs arise at the c-MYC enhancer in human BC cells, and their defective repair changes the activation profile of enhancers and induces the overexpression of many genes, including the c-MYC oncogene. CRISPR/Cas9 cleavage at the enhancer also causes c-MYC overexpression, indicating that this DSB causes c-MYC overexpression. Estrogen treatment induced c-Myc protein overexpression in mammary epithelial cells of ATM-deficient mice. In conclusion, ATM suppresses the c-Myc-driven proliferative effects of estrogens, possibly explaining such tissue-specific oncogenesis.

Design of Molecules with Low Hole and Electron Reorganization Energy Using DFT Calculations and Bayesian Optimization.

Materials exhibiting higher mobility than conventional organic semiconducting materials, such as fullerenes and fused thiophenes, are in high demand for applications in printed electronics. To discover new molecules that might show improved charge mobility, the adaptive design of experiments (DoE) to design molecules with low reorganization energy was performed by combining density functional theory (DFT) methods and machine learning techniques. DFT-calculated values of 165 molecules were used as an initial training dataset for a Gaussian process regression (GPR) model, and five rounds of molecular designs applying the GPR model and validation via DFT calculations were executed. As a result, new molecules whose reorganization energy is smaller than the lowest value in the initial training dataset were successfully discovered.

Pilot-scale extraction of polyphenols from spent black tea by semi-continuous subcritical solvent extraction.

Spent black tea (SBT) is a residue from tea beverage production and considered as a potential source of active polyphenols. This study aimed to develop a pilot-scale process on semi-continuous subcritical solvent extraction (SSE) of polyphenols from SBT by exploiting the lab-scale knowledge. Treatment of SBT with ethanol-water (50% w/w) as solvent at 125 °C and 0.3 MPa achieved a significantly higher yield of polyphenols (80.82 g gallic acid equivalents/kg black tea) with antioxidant activity (64.20 g gallic acid equivalents/kg black tea), compared to hot water extraction (HWE). SSE increased the soluble matter content in extracts than HWE. Based on the results of LC-MS, theaflavin-3,3'-digallate was the most abundant polyphenol from a total of 12 compounds to be extracted by SBT with 50% ethanol. The results suggested that SSE can be used as a scale-up extraction method to recover polyphenols from SBT.

XRCC1 counteracts poly(ADP ribose)polymerase (PARP) poisons, olaparib and talazoparib, and a clinical alkylating agent, temozolomide, by promoting the removal of trapped PARP1 from broken DNA.

Base excision repair (BER) removes damaged bases by generating single-strand breaks (SSBs), gap-filling by DNA polymerase ß (POLß), and resealing SSBs. A base-damaging agent, methyl methanesulfonate (MMS) is widely used to study BER. BER increases cellular tolerance to MMS, anti-cancer base-damaging drugs, temozolomide, carmustine, and lomustine, and to clinical poly(ADP ribose)polymerase (PARP) poisons, olaparib and talazoparib. The poisons stabilize PARP1/SSB complexes, inhibiting access of BER factors to SSBs. PARP1 and XRCC1 collaboratively promote SSB resealing by recruiting POLß to SSBs, but XRCC1-/- cells are much more sensitive to MMS than PARP1-/- cells. We recently report that the PARP1 loss in XRCC1-/- cells restores their MMS tolerance and conclude that XPCC1 facilitates the release of PARP1 from SSBs by maintaining its autoPARylation. We here show that the PARP1 loss in XRCC1-/- cells also restores their tolerance to the three anti-cancer base-damaging drugs, although they and MMS induce different sets of base damage. We reveal the synthetic lethality of the XRCC1-/- mutation, but not POLß-/- , with olaparib and talazoparib, indicating that XRCC1 is a unique BER factor in suppressing toxic PARP1/SSB complex and can suppress even when PARP1 catalysis is inhibited. In conclusion, XRCC1 suppresses the PARP1/SSB complex via PARP1 catalysis-dependent and independent mechanisms.

Repair pathways for radiation DNA damage under normoxic and hypoxic conditions: Assessment with a panel of repair-deficient human TK6 cells.

Various types of DNA lesions are produced when cells are exposed to ionizing radiation (IR). The type and yield of IR-induced DNA damage is influenced by the oxygen concentration. Thus, different DNA repair mechanisms may be involved in the response of normoxic and hypoxic cells to irradiation with IR. However, differences between the repair mechanisms of IR-induced DNA damage under normoxic versus hypoxic conditions have not been clarified. Elucidating the relative contribution of individual repair factors to cell survival would give insight into the repair mechanisms operating in irradiated normoxic and hypoxic cells. In the present study, we used a panel of repair-deficient human TK6 cell lines that covered seven repair pathways. Cells were irradiated with X-rays under normoxic and hypoxic conditions, and the sensitivities of each mutant relative to the wild-type (i.e. relative sensitivity) were determined for normoxic and hypoxic conditions. The sensitivity of cells varied depending on the type of repair defects. However, for each repair mutant, the relative sensitivity under normoxic conditions was comparable to that under hypoxic conditions. This result indicates that the relative contribution of individual repair pathways to cell survival is comparable in normoxic and hypoxic cells, although the spectrum of IR-induced DNA damage in hypoxic cells differs from that of normoxic cells.

Development and Characterization of Functional Starch-Based Films Incorporating Free or Microencapsulated Spent Black Tea Extract.

Antioxidant polyphenols in black tea residue are an underused source of bioactive compounds. Microencapsulation can turn them into a valuable functional ingredient for different food applications. This study investigated the potential of using spent black tea extract (SBT) as an active ingredient in food packaging. Free or microencapsulated forms of SBT, using a pectin-sodium caseinate mixture as a wall material, were incorporated in a cassava starch matrix and films developed by casting. The effect of incorporating SBT at different polyphenol contents (0.17% and 0.34%) on the structural, physical, and antioxidant properties of the films, the migration of active compounds into different food simulants and their performance at preventing lipid oxidation were evaluated. The results showed that adding free SBT modified the film structure by forming hydrogen bonds with starch, creating a less elastic film with antioxidant activity (173 and 587 µg(GAE)/g film). Incorporating microencapsulated SBT improved the mechanical properties of active films and preserved their antioxidant activity (276 and 627 µg(GAE)/g film). Encapsulates significantly enhanced the release of antioxidant polyphenols into both aqueous and fatty food simulants. Both types of active film exhibited better barrier properties against UV light and water vapour than the control starch film and delayed lipid oxidation up to 35 d. This study revealed that starch film incorporating microencapsulated SBT can be used as a functional food packaging to protect fatty foods from oxidation.

Valorization of spent black tea by recovery of antioxidant polyphenolic compounds: Subcritical solvent extraction and microencapsulation.

Spent black tea (SBT), waste remaining after producing tea beverages, is potentially an underutilized source of antioxidant phenolic compounds. This study evaluated the integrated processes of subcritical solvent extraction of polyphenols from SBT followed by microencapsulation to improve the stability of obtained extract. Optimization of extraction conditions was carried out by response surface methodology for the best recovery of antioxidant phenolic compounds. Two variables [temperature (°C) and ethanol concentration (%)] were used to design the optimization model using central composite inscribed. Extraction temperature of 180°C and ethanol concentration of 71% were optimal for the highest yield of total polyphenols (126.89 mg gallic acid equiv./g SBT) and 2,2-diphenyl-1-picrylhydrazyl scavenging activity (69.08 mg gallic acid equiv./g SBT). The extract was encapsulated using pectin, sodium caseinate, and a blend of these compounds (ratio 1:1) as wall materials by spray drying. The wall material significantly influenced (p < .05) encapsulation efficiency, particle size, morphology, thermal stability, crystallinity, and storage stability. The blend of wall materials produced an amorphous powder with the highest phenolic retention (94.28%) in the accelerated storage at 45°C for 40 days. The microcapsules prepared with sodium caseinate were smaller with lowest mean diameter and highest thermal stability than the other types of materials. Obtained microencapsulates have potential use in different food systems to enhance their antioxidant property.

Biogas production management systems with model predictive control of anaerobic digestion processes.

We developed a biogas production management system to control biogas production by determining the feedstock inputs to the anaerobic digestion process according to fluctuations of the renewable energy supply. The developed system consists of three functions: a prediction model for the anaerobic digestion processes, a parameter-estimation system, and a feedstock-determination controller. A prediction model for the anaerobic digestion processes in a state-space representation was constructed for the input-output relationship of biogas generation from organic compounds and the state of methane fermentation. A parameter-estimation system that estimated the parameters included in the prediction model from actual operating process data was built based on adaptive identification theory. The feedstock-determination controller was established based on model predictive control as a method to control biogas production. From the results of the identification experiment, the least square estimator of the parameters converged as the training data increased, and a reliable parameter was given in 1 week. From the results of the numerical simulation and the control experiment, it was confirmed that the biogas production management system developed in this study had a high prediction accuracy and control performance.

Topoisomerase I-driven repair of UV-induced damage in NER-deficient cells.

Nucleotide excision repair (NER) removes helix-destabilizing adducts including ultraviolet (UV) lesions, cyclobutane pyrimidine dimers (CPDs), and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). In comparison with CPDs, 6-4PPs have greater cytotoxicity and more strongly destabilizing properties of the DNA helix. It is generally believed that NER is the only DNA repair pathway that removes the UV lesions as evidenced by the previous data since no repair of UV lesions was detected in NER-deficient skin fibroblasts. Topoisomerase I (TOP1) constantly creates transient single-strand breaks (SSBs) releasing the torsional stress in genomic duplex DNA. Stalled TOP1-SSB complexes can form near DNA lesions including abasic sites and ribonucleotides embedded in chromosomal DNA. Here we show that base excision repair (BER) increases cellular tolerance to UV independently of NER in cancer cells. UV lesions irreversibly trap stable TOP1-SSB complexes near the UV damage in NER-deficient cells, and the resulting SSBs activate BER. Biochemical experiments show that 6-4PPs efficiently induce stable TOP1-SSB complexes, and the long-patch repair synthesis of BER removes 6-4PPs downstream of the SSB. Furthermore, NER-deficient cancer cell lines remove 6-4PPs within 24 h, but not CPDs, and the removal correlates with TOP1 expression. NER-deficient skin fibroblasts weakly express TOP1 and show no detectable repair of 6-4PPs. Remarkably, the ectopic expression of TOP1 in these fibroblasts led them to completely repair 6-4PPs within 24 h. In conclusion, we reveal a DNA repair pathway initiated by TOP1, which significantly contributes to cellular tolerance to UV-induced lesions particularly in malignant cancer cells overexpressing TOP1.

Effect of temperature on the hydrolysis of levan treated with compressed hot water fluids.

The hydrolysis of levan using compressed hot water for the production of functional fructooligosaccharides (FOSs) was investigated. Levans from Erwinia herbicola (EH) and Halomonas smyrnensis (HS) were characterized using scanning electron microscopy and light scattering techniques, and hydrolyzed using compressed hot water at four temperatures (120, 140, 160, and 180°C). The hydrolysates were analyzed using high-performance liquid chromatography and electrospray ionization-mass spectrometry. Levan HS showed a crystalline morphology, whereas levan EH showed an aggregated structure. Both levans had molar masses on the order of 106 g/mol, but levan EH had a smaller radius of gyration, hydrodynamic radius, and intrinsic viscosity. Levan EH hydrolyzed into FOSs at approximately 120°C, whereas levan HS required a temperature of at least 160°C, possibly because of differences in the degree of branching of the two levans. Both samples were degraded to fructose when treated at 180°C.

Parboiled Paddy Drying with Different Dryers: Thermodynamic and Quality Properties, Mathematical Modeling Using ANNs Assessment.

The effect of hybrid infrared-convective (IRC), microwave (MIC) and infrared-convective-microwave (IRCM) drying methods on thermodynamic (drying kinetics, effective moisture diffusivity coefficient (Deff), specific energy consumption (SEC)) and quality (head rice yield (HRY), color value and lightness) characteristics of parboiled rice samples were investigated in this study. Experimental data were fitted into empirical drying models to explain moisture ratio (MR) variations during drying. The Artificial Neural Network (ANN) method was applied to predict MR. The IRCM method provided shorter drying time (reduce percentage = 71%) than IRC (41%) and microwave (69%) methods. The Deff of MIC drying (6.85 × 10-11-4.32 × 10-10 m2/s) was found to be more than the observed in IRC (1.32 × 10-10-1.87 × 10-10 m2/s) and IRCM methods (1.58 × 10-11-2.31 × 10-11 m2/s). SEC decreased during drying. Microwave drying had the lowest SEC (0.457 MJ/kg) compared to other drying methods (with mean 28 MJ/kg). Aghbashlo's model was found to be the best for MR prediction. According to the ANN results, the highest determination coefficient (R2) values for MR prediction in IRC, IRCM and MIC drying methods were 0.9993, 0.9995 and 0.9990, respectively. The HRY (from 60.2 to 74.07%) and the color value (from 18.08 to 19.63) increased with the drying process severity, thereby decreasing the lightness (from 57.74 to 62.17). The results of this research can be recommended for the selection of the best dryer for parboiled paddy. Best drying conditions in the study is related to the lowest dryer SEC and sample color value and the highest HRY and sample lightness.

Adaptive identification of anaerobic digestion process for biogas production management systems.

To achieve the goals of sustainable development, supplies of renewable energy must be increased and methods of stable production developed. This study focused on the anaerobic digestion process using biomass as a raw material, which represents a renewable energy resource which is robust to environmental change and can be adjusted to suit supply and demand. A state-space model of the process was built in this study, consisting of two differential equations and one algebraic equation. The parameters included in the model are dependent on the operating conditions of the process. Automatic estimation of parameters from the input and output data of the process enables easy use of the model under any operating conditions. An adaptive-identifier control system was introduced as the parameter-estimation system, which made it possible to obtain the least squares estimate of parameters. When accumulated biogas generation per day was predicted using the model, goodness-of-fit analysis indicated an accuracy of over 80% in all cases, validating the model and estimated parameters. Future tasks will involve implementation of model predictive control into anaerobic digestion processes with the model and parameter-estimation system developed in this study.

Motion analysis of operating a balance exercise assist robot system during forward and backward movements.

[Purpose] Stand-and-ride personal mobility devices controlled by movements of the user's center of gravity are used for balance training. We aimed to describe the physical activity required to operate this type of mobility device. [Participants and Methods] Eleven healthy males performed the following tasks: 1) moving their center of gravity forward or backward while standing on the floor (control task) and, 2) moving the mobility device forward or backward by moving their center of gravity (experimental task). [Results] We observed that the displacement of the center of gravity and the center of pressure, as well as angular displacements of the hips and knee joints, and maximum muscle activities of the biceps femoris, the medial head of the gastrocnemius and peroneus longus muscles were lesser during the experimental than during the control task. The distance moved by the device was significantly greater than the displacement of the user's center of gravity during the experimental task. [Conclusion] We observed that moving the device forward or backward required lesser physical activity than that required to shift the user's center of gravity forward or backward while standing on the floor. Additionally, we observed that even a small displacement of the user's center of gravity produced a large displacement of the device. We concluded that during balance training, the greater and more easily perceived movement of the mobility device would provide helpful feedback to the user.

Kinematic, kinetic, and electromyographic characteristics during quiet standing on a balance exercise assist robot.

[Purpose] The balance exercise assist robot is a training device based on a personal transport assistance robot ridden in the standing position. The personal transport assistance robot uses an inverted pendulum control system and moves in response to movements of the user's center of gravity. The purpose of this study was to describe the characteristics of postural control during the action of stopping the personal transport assistance robot. [Participants and Methods] Eleven healthy male participants were required to maintain a standing position for 30 s; each task was performed 10 times. The measurement conditions were as follows: (1) on the floor; (2) on the robot, touching the handlebars; and (3) on the robot, not touching the handlebars. [Results] During the robotic tasks, the total locus lengths of the center of gravity and total joint momentums of the hip, knee, and ankle joints were larger, and the amount of displacement of the center of pressure was smaller than that during the floor task. Posture control on the robot was performed actively by mechanical interaction of the ankle, knee, and hip joints within a small base of support. [Conclusion] The balance exercise assist robot can be useful for postural control exercises because maintaining a standing position on the personal transport assistance robot required active postural control.

ALC1/CHD1L, a chromatin-remodeling enzyme, is required for efficient base excision repair.

ALC1/CHD1L is a member of the SNF2 superfamily of ATPases carrying a macrodomain that binds poly(ADP-ribose). Poly(ADP-ribose) polymerase (PARP) 1 and 2 synthesize poly(ADP-ribose) at DNA-strand cleavage sites, promoting base excision repair (BER). Although depletion of ALC1 causes increased sensitivity to various DNA-damaging agents (H2O2, UV, and phleomycin), the role played by ALC1 in BER has not yet been established. To explore this role, as well as the role of ALC1's ATPase activity in BER, we disrupted the ALC1 gene and inserted the ATPase-dead (E165Q) mutation into the ALC1 gene in chicken DT40 cells, which do not express PARP2. The resulting ALC1-/- and ALC1-/E165Q cells displayed an indistinguishable hypersensitivity to methylmethane sulfonate (MMS), an alkylating agent, and to H2O2, indicating that ATPase plays an essential role in the DNA-damage response. PARP1-/- and ALC1-/-/PARP1-/- cells exhibited a very similar sensitivity to MMS, suggesting that ALC1 and PARP1 collaborate in BER. Following pulse-exposure to H2O2, PARP1-/- and ALC1-/-/PARP1-/- cells showed similarly delayed kinetics in the repair of single-strand breaks, which arise as BER intermediates. To ascertain ALC1's role in BER in mammalian cells, we disrupted the ALC1 gene in human TK6 cells. Following exposure to MMS and to H2O2, the ALC1-/- TK6 cell line showed a delay in single-strand-break repair. We therefore conclude that ALC1 plays a role in BER. Following exposure to H2O2, ALC1-/- cells showed compromised chromatin relaxation. We thus propose that ALC1 is a unique BER factor that functions in a chromatin context, most likely as a chromatin-remodeling enzyme.