Morphological growth dynamics, mechanical stability, and active microtubule mechanics underlying spindle self-organization.

The spindle is a dynamic intracellular structure self-organized from microtubules and microtubule-associated proteins. The spindle's bipolar morphology is essential for the faithful segregation of chromosomes during cell division, and it is robustly maintained by multifaceted mechanisms. However, abnormally shaped spindles, such as multipolar spindles, can stochastically arise in a cell population and cause chromosome segregation errors. The physical basis of how microtubules fail in bipolarization and occasionally favor nonbipolar assembly is poorly understood. Here, using live fluorescence imaging and quantitative shape analysis in Xenopus egg extracts, we find that spindles of varied shape morphologies emerge through nonrandom, bistable self-organization paths, one leading to a bipolar and the other leading to a multipolar phenotype. The bistability defines the spindle's unique morphological growth dynamics linked to each shape phenotype and can be promoted by a locally distorted microtubule flow that arises within premature structures. We also find that bipolar and multipolar spindles are stable at the steady-state in bulk but can infrequently switch between the two phenotypes. Our microneedle-based physical manipulation further demonstrates that a transient force perturbation applied near the assembled pole can trigger the phenotypic switching, revealing the mechanical plasticity of the spindle. Together with molecular perturbation of kinesin-5 and augmin, our data propose the physical and molecular bases underlying the emergence of spindle-shape variation, which influences chromosome segregation fidelity during cell division.

Gre factors prevent thermal and mechanical stresses induced by terahertz irradiation during transcription.

During transcription in cells, the transcription complex consisting of RNA polymerase, DNA and nascent RNA is exposed to fluctuating temperature and pressure. However, little is known about the mechanism of transcriptional homeostasis under fluctuating physical parameters. In this study, we generated these fluctuating parameters using pulsed local heating and acoustic waves in the reaction system of transcription by Escherichia coli RNA polymerase, using a terahertz free-electron laser. We demonstrated that transcription processes, including abortive initiation and elongation pausing, and the fidelity of elongation are significantly affected by the laser-based local perturbations. We also found that all these functional alternations in the transcription process are almost completely mitigated by the presence of Gre proteins. It is well known that Gre proteins enhance RNA cleavage of polymerase by binding to the pore structure termed secondary channel. Recently, the chaperone activities have also been proposed for Gre proteins, yet the details directly associated with transcription are largely unknown. Our finding indicates that Gre proteins are necessary for maintaining transcriptional homeostasis under thermal and mechanical stresses.

Ultrafast time-resolved single-shot birefringence microscopy for laser-induced anisotropy.

The interaction between ultrashort laser pulses and materials in the ultrafast time domain, especially regarding the effect of laser polarization, has attracted much attention. In this study, ultrafast time-resolved single-shot birefringence microscopy is performed to observe laser-induced anisotropy. The birefringences of the optical Kerr effect and laser-induced anisotropic nanostructures by femtosecond laser pulses in silica glass are measured, and their slow axis is confirmed to correspond to the linear polarization angle of the pump light. We discuss the time variations of these birefringences in the picosecond time domain.

Nonthermal excitation effects mediated by sub-terahertz radiation on hydrogen exchange in ubiquitin.

Water dynamics in the hydration layers of biomolecules play crucial roles in a wide range of biological functions. A hydrated protein contains multiple components of diffusional and vibrational dynamics of water and protein, which may be coupled at ∼0.1-THz frequency (10-ps timescale) at room temperature. However, the microscopic description of biomolecular functions based on various modes of protein-water-coupled motions remains elusive. A novel approach for perturbing the hydration dynamics in the subterahertz frequency range and probing them at the atomic level is therefore warranted. In this study, we investigated the effect of klystron-based, intense 0.1-THz excitation on the slow dynamics of ubiquitin using NMR-based measurements of hydrogen-deuterium exchange. We demonstrated that the subterahertz irradiation accelerated the hydrogen-deuterium exchange of the amides located in the interior of the protein and hydrophobic surfaces while decelerating this exchange in the amides located in the surface loop and short 310 helix regions. This subterahertz-radiation-induced effect was qualitatively contradictory to the increased-temperature-induced effect. Our results suggest that the heterogeneous water dynamics occurring at the protein-water interface include components that are nonthermally excited by the subterahertz radiation. Such subterahertz-excited components may be linked to the slow function-related dynamics of the protein.

Independent contribution of optical attenuation length in ultrafast laser-induced structural change.

Although laser irradiation with femtosecond pulses is known to generate crystallization and morphological changes, the contribution of optical parameters to material changes is still in discussion. Here, we compare two structures irradiated near Si-L2,3 edges by an extreme ultraviolet femtosecond pulse. Our result implies that, despite the femtosecond irradiation regime, these values of the optical attenuation length between the wavelengths of 10.3-nm and 13.5-nm differ by one order of magnitude. From the structural comparison, the original crystalline state was maintained upon irradiation at 13.5-nm, on the other hand, transition to an amorphous state occurred at 10.3-nm. The difference in optical attenuation length directly influence to the decision of material crystallization or morphological changes, even if the irradiation condition is under the femtosecond regime and same pulse duration. Our result reveals the contribution of optical attenuation length in ultrafast laser-induced structural change.

LC-MS/MS and chiroptical spectroscopic analyses of multidimensional metabolic systems of chiral thalidomide and its derivatives.

Enantiomeric thalidomide undergoes various kinds of biotransformations including chiral inversion, hydrolysis, and enzymatic oxidation, which results in several metabolites, thereby adding to the complexity in the understanding of the nature of thalidomide. To decipher this complexity, we analyzed the multidimensional metabolic reaction networks of thalidomide and related molecules in vitro. Characteristic patterns in the amount of various metabolites of thalidomide and related molecules generated during a combination of chiral inversion, hydrolysis, and hydroxylation were observed using liquid chromatography-tandem mass spectrometry and chiroptical spectroscopy. We found that monosubstituted thalidomide derivatives exhibited different time-dependent metabolic patterns compared with thalidomide. We also revealed that monohydrolyzed and monohydroxylated metabolites of thalidomide were likely to generate mainly by a C-5 oxidation of thalidomide and subsequent ring opening of the hydroxylated metabolite. Since chirality was conserved in most of these metabolites during metabolism, they had the same chirality as that of nonmetabolized thalidomide. Our findings will contribute toward understanding the significant pharmacological effects of the multiple metabolites of thalidomide and its derivatives.

[Clinical Analysis of Combination Chemotherapy Using High Dose Methotrexate, Rituximab, and Vincristine with or without Procarbazine for Elderly Patients with Diffuse Large B-Cell Lymphoma of the Central Nervous System].

We studied the clinical effects of high-dose methotrexate(HD-MTX)combined with rituximab and vincristine in 5 elderly patients, aged 65-83 years, with diffuse large B-cell lymphoma of the central nervous system(DLBCL CNS). Patients aged 65- 71 years were given 3.0 g/m2 of HD-MTX, while patients aged 75-83 years were given 1.5 g/m2 of the drug. All patients showed responses; 1 CR and 1 PR in MTX 3.0 g/m2 group, and 2 CRs and 1 PR in MTX 1.5 g/m2 group.

Optical Activity and Optical Anisotropy in Photomechanical Crystals of Chiral Salicylidenephenylethylamines.

Introducing chirality into photomechanical crystals is beneficial for the diversification of mechanical motion. Measurement of the chiroptical and optical anisotropic properties of chiral crystals is indispensable for evaluating photomechanical crystals. The platelike crystals of S- and R-enantiomers of photochromic N-3,5-di-tert-butylsalicylidene-1-phenylethylamine in enol form (enol-(S)-1 and enol-(R)-1) caused bending motion with twisting upon ultraviolet (UV) light irradiation, due to shrinkage along the length and width directions of the irradiated surface, based on the optimized crystal structure of the photoisomerized trans-keto-(S)-1. By employing the generalized high-accuracy universal polarimeter (G-HAUP), optical anisotropic (linear birefringence, LB; linear dichroism, LD) as well as chiroptical (circular birefringence, CB; circular dichroism, CD) spectra of both the enantiomeric crystals on the (001) face were simultaneously measured before and under continuous UV irradiation. The LD peak was observed at 330 nm in the negative sign, derived from the π-π* transition of the intramolecularly hydrogen-bonded salicylidenimino moiety. The CD spectra of the S and R crystals revealed the negative and positive Cotton effect at 330 nm, respectively, and new peaks appeared at 460 nm under UV light irradiation due to photoisomerization to the S and R trans-keto isomers at around 10% conversion. The CB and CD spectra evaluated by the HAUP measurement were opposite to those measured in the hexane solution, as well as those simulated by quantum chemical calculation. The dissymmetry parameter, g, of the enol-(S)-1 crystal along the c axis (0.013) was approximately 10 times larger than the g values in the solution (0.0010) and by calculation (0.0016).

PIGB maintains nuclear lamina organization in skeletal muscle of Drosophila.

The nuclear lamina (NL) plays various roles and participates in nuclear integrity, chromatin organization, and transcriptional regulation. Lamin proteins, the main components of the NL, form a homogeneous meshwork structure under the nuclear envelope. Lamins are essential, but it is unknown whether their homogeneous distribution is important for nuclear function. Here, we found that PIGB, an enzyme involved in glycosylphosphatidylinositol (GPI) synthesis, is responsible for the homogeneous lamin meshwork in Drosophila. Loss of PIGB resulted in heterogeneous distributions of B-type lamin and lamin-binding proteins in larval muscles. These phenotypes were rescued by expression of PIGB lacking GPI synthesis activity. The PIGB mutant exhibited changes in lamina-associated domains that are large heterochromatic genomic regions in the NL, reduction of nuclear stiffness, and deformation of muscle fibers. These results suggest that PIGB maintains the homogeneous meshwork of the NL, which may be essential for chromatin distribution and nuclear mechanical properties.

Characteristic oxygen K-edge circular dichroism spectra of amino acid films by improved measurement technique.

Circular dichroism (CD) spectroscopy in the soft x-ray energy region is a new tool to study the local structure of chiral materials. In this paper, we introduce a method to measure high-quality CD spectra in the oxygen K-edge energy region. Characteristic CD spectra of thin films of the amino acids L-tyrosine and L-aspartic acid are reported and compared with those of films of L-alanine and L-serine. The signals from the oxygen 1s → π∗ transitions of COO-, which is a common moiety in these amino acids, reflect the local geometry of each amino acid.

Nonthermal acceleration of protein hydration by sub-terahertz irradiation.

The collective intermolecular dynamics of protein and water molecules, which overlap in the sub-terahertz (THz) frequency region, are relevant for expressing protein functions but remain largely unknown. This study used dielectric relaxation (DR) measurements to investigate how externally applied sub-THz electromagnetic fields perturb the rapid collective dynamics and influence the considerably slower chemical processes in protein-water systems. We analyzed an aqueous lysozyme solution, whose hydration is not thermally equilibrated. By detecting time-lapse differences in microwave DR, we demonstrated that sub-THz irradiation gradually decreases the dielectric permittivity of the lysozyme solution by reducing the orientational polarization of water molecules. Comprehensive analysis combining THz and nuclear magnetic resonance spectroscopies suggested that the gradual decrease in the dielectric permittivity is not induced by heating but is due to a slow shift toward the hydrophobic hydration structure in lysozyme. Our findings can be used to investigate hydration-mediated protein functions based on sub-THz irradiation.

Determination of nitrite, nitrate, bromide, and iodide in seawater by ion chromatography with UV detection using dilauryldimethylammonium-coated monolithic ODS columns and sodium chloride as an eluent.

A method was developed for determination of inorganic anions, including nitrite (NO(2)(-)), nitrate (NO(3)(-)), bromide (Br(-)), and iodide (I(-)), in seawater by ion chromatography (IC). The IC system used two dilauryldimethylammonium bromide (DDAB)-coated monolithic ODS columns (50 × 4.6 mm i.d. and 100 × 4.6 mm i.d.) connected in series for separation of the ions. Aqueous NaCl (0.5 mol/L; flow rate, 3 mL/min) containing 5 mmol/L phosphate buffer (pH 5) was used as the eluent, and detection was with a UV detector at 225 nm. The monolithic ODS columns were coated and equilibrated with a 1-mmol/L DDAB solution (in H(2)O/methanol, 90:10 v/v). The hydrophilic ions (NO(2)(-), NO(3)(-), and Br(-)) were separated within 3 min and the retention time of I(-) was 16 min. No interferences from matrix ions, such as chloride and sulfate ions, were observed in 35 ‰ artificial seawater. The detection limits were 0.6 µg/L for NO(2)(-), 1.1 µg/L for NO(3)(-), 70 µg/L for Br(-), and 1.6 µg/L for I(-) with a 200-µL sample injection. The performance of the coated columns was maintained without addition of DDAB in the eluent. The IC system was successfully applied to real seawater samples with recovery rates of 94-108 % for all ions.

Dynamics of Actin Cytoskeleton and Their Signaling Pathways during Cellular Wound Repair.

The repair of wounded cell membranes is essential for cell survival. Upon wounding, actin transiently accumulates at the wound site. The loss of actin accumulation leads to cell death. The mechanism by which actin accumulates at the wound site, the types of actin-related proteins participating in the actin remodeling, and their signaling pathways are unclear. We firstly examined how actin accumulates at a wound site in Dictyostelium cells. Actin assembled de novo at the wound site, independent of cortical flow. Next, we searched for actin- and signal-related proteins targeting the wound site. Fourteen of the examined proteins transiently accumulated at different times. Thirdly, we performed functional analyses using gene knockout mutants or specific inhibitors. Rac, WASP, formin, the Arp2/3 complex, profilin, and coronin contribute to the actin dynamics. Finally, we found that multiple signaling pathways related to TORC2, the Elmo/Doc complex, PIP2-derived products, PLA2, and calmodulin are involved in the actin dynamics for wound repair.

Neutron flat-panel detector using In-Ga-Zn-O thin-film transistor.

Neutron imaging is a powerful tool for observing the internal structure of an object without destroying the object. Neutron imaging (neutron radiography) is a prominent application of neutrons but still requires significant improvements, for example, in sensitivity, resolution, radiation hardness, and handling of neutron imaging detectors. This paper presents the development and the first neutron imaging results of a neutron flat-panel detector (nFPD) based on an In-Ga-Zn-O (IGZO) thin-film transistor (TFT)/photodiode array coupled with a LiF/ZnS scintillator sheet. Direct photo-coupling to the scintillator increases the light collection efficiency. Moreover, unlike lens-coupled neutron cameras, the proposed detector is compact and easy to handle. Owing to the high off-state resistance of IGZO TFTs, their leakage current is lower than that of conventional TFTs, enabling the IGZO TFTs to hold an accumulated charge for a longer period of time and allowing longer exposure times for imaging. This would be a powerful feature for imaging at compact neutron sources with limited flux. This paper reports on the first neutron imaging results with an IGZO nFPD, its performance evaluation, and a demonstration of three-dimensional computed tomography with neutrons.

Local body weight measurement of the spindle.

The spindle is a micron-sized chromosome segregation machine built from microtubules and many other proteins. In this issue of Developmental Cell, Biswas et al. (2021) use sophisticated imaging and Xenopus egg extracts to show that the spindle's mass density is only as much as the surrounding cytoplasm, contrary to popular belief.

A 'dynamic adder model' for cell size homeostasis in Dictyostelium cells.

After a cell divides into two daughter cells, the total cell surface area of the daughter cells should increase to the original size to maintain cell size homeostasis in a single cell cycle. Previously, three models have been proposed to explain the regulation of cell size homeostasis: sizer, timer, and adder models. Here, we precisely measured the total cell surface area of Dictyostelium cells in a whole cell cycle by using the agar-overlay method, which eliminated the influence of surface membrane reservoirs, such as microvilli and membrane wrinkles. The total cell surface area exponentially increased during interphase, slightly decreased at metaphase, and then increased by approximately 20% during cytokinesis. From the analysis of the added surface area, we concluded that the cell size was regulated by the adder or near-adder model in interphase. This adder model is not caused by a simple cell membrane addition, but is more dynamic due to the rapid cell membrane turnover. We propose a 'dynamic adder model' to explain cell size homeostasis in interphase.

Vaginal temperature before calving assessed with wireless vaginal temperature sensor in dairy and beef cattle.

We evaluated the daily and hourly vaginal temperature changes and the relationships between the dams' breed and parity by using a commercially available vaginal temperature sensor in 72 Holstein (Hol) calvings and 101 Japanese Black (JB) calvings. Vaginal temperature sensors inserted 7-10 days before the expected calving day sounded two alerts: when the temperature fell below the threshold (Alert 1), and when the sensor reached the ambient temperature after falling out of the dam's vagina with the rupture of the allantoic sac (Alert 2). The durations from Alert 1 to Alert 2 (Time 1) and from Alert 2 to delivery (Time 2) were calculated. Only Time 1 in the Hol group tended to be affected by parity and parity × calf body weight. In the JB group, none of the factors examined affected Time 1 or Time 2. The alert detection rates did not differ by parity in either breed or by the temperature threshold in Hol. However, the Hol group's alert detection rate was significantly lower than the JB group's (p < 0.05). The daily average temperature was higher in the Hol group and the primiparous dams than those in the JB and multiparous dams; it increased slightly from Day -7 to -3 (Day 0 = the day of calving) and then dropped dramatically on Days -1 and 0. The hourly vaginal temperature difference from -48 h of calving showed a typical pattern, i.e., a decrease from -30 h of Alert 1 and an increase at -6 h of Alert 1. The decrease and increase might be the regression of the pregnant corpus luteum and the beginning of the contractions, respectively. The temperature differences were significantly affected by parity and calving ease (p < 0.01). The primiparous dams showed wider temperature differences compared to the multiparous dams in both breeds (p < 0.001). No typical temperature difference pattern was observed in assisted calving or dystocia. The alert detection rate, the Time durations, and the vaginal temperature differences were affected by the dams' breed and parity. However, measuring vaginal temperatures proved useful for predicting the calving regardless of the breed and parity. The effect of calving ease remains unclear due to the low number of assisted calvings herein.

RanGTP and the actin cytoskeleton keep paternal and maternal chromosomes apart during fertilization.

Zygotes require two accurate sets of parental chromosomes, one each from the mother and the father, to undergo normal embryogenesis. However, upon egg-sperm fusion in vertebrates, the zygote has three sets of chromosomes, one from the sperm and two from the egg. The zygote therefore eliminates one set of maternal chromosomes (but not the paternal chromosomes) into the polar body through meiosis, but how the paternal chromosomes are protected from maternal meiosis has been unclear. Here we report that RanGTP and F-actin dynamics prevent egg-sperm fusion in proximity to maternal chromosomes. RanGTP prevents the localization of Juno and CD9, egg membrane proteins that mediate sperm fusion, at the cell surface in proximity to maternal chromosomes. Following egg-sperm fusion, F-actin keeps paternal chromosomes away from maternal chromosomes. Disruption of these mechanisms causes the elimination of paternal chromosomes during maternal meiosis. This study reveals a novel critical mechanism that prevents aneuploidy in zygotes.

Application of magnetic Compton scattering for spin-specific magnetic hysteresis measurement.

An application of magnetic Compton scattering as a new tool to measure a spin-specific magnetic hysteresis (SSMH) loop is introduced and its validity demonstrated. The applied magnetic field dependence of the integrated intensity of magnetic Compton scattering spectra, which reflect only the spin-dependent magnetic properties of magnetically active electrons, was interpreted as the spin-specific hysteresis. The spin magnetization of amorphous Tb(33)Co(67) film was observed and its SSMH loop exhibited qualitative agreement with the ordinal magnetic hysteresis loop measured using a conventional vibrating sample magnetometer.